Antiviral phosphonate analogs

ABSTRACT

The invention is related to phosphorus substituted compounds with antiviral activity, compositions containing such compounds, and therapeutic methods that include the administration of such compounds, as well as to processes and intermediates useful for preparing such compounds.

PRIORITY OF INVENTION

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Patent Application Ser. Nos. 60/465,630, 60/465,400,60/465,587, 60/465,463, 60/465,602, 60/465,598, 60/465,633, 60/465,550,60/465,610, 60/465,720, 60/465,634, 60/465,537, 60/465,698, 60/465,667,60/465,554, 60/465,553, 60/465,561, 60/465,548, 60/465,696, 60/465,347,60/465,289, 60/465,478, 60/465,600, 60/465,591, 60/465,684, 60/465,821,60/465,647, 60/465,742, 60/465,649, 60/465,690, 60/465,469, 60/465,408,60/465,608, 60/465,584, 60/465,687, 60/465,759, 60/465,559, 60/465,322,60/465,377, 60/465,844, and 60/465,544, all filed Apr. 25, 2003; and toU.S. Provisional Patent Application Ser. No. 60/490,799, filed Jul. 29,2003; and to U.S. Provisional Patent Application Ser. Nos. 60/495,687,60/495,490, 60/495,805, 60/495,684, 60/495,600, 60/495,342, 60/495,564,60/495,772, 60/495,592, 60/495,453, 60/495,491, 60/495,964, 60/495,317,60/495,696, 60/495,760, 60/495,334, 60/495,671, 60/495,349, 60/495,273,60/495,763, 60/495,345, 60/495,602, 60/495,343, 60/495,344, 60/495,278,60/495,277, 60/495,275, 60/495,630, 60/495,485, 60/495,430, 60/495,388,60/495,341, 60/495,631, 60/495,633, 60/495,632, 60/495,539, 60/495,387,60/495,392, 60/495,425, 60/495,393, and 60/495,616, all filed Aug. 15,2003; and to U.S. Provisional Patent Application Ser. No. 60/510,245,filed Oct. 10, 2003; and to U.S. Provisional Patent Application Ser.Nos. 60/514,202, 60/513,948, and 60/514,258, all filed Oct. 24, 2003;and to U.S. Provisional Patent Application Ser. No. 60/515,266, filedOct. 29, 2003; and to U.S. Provisional Patent Application Ser. No.60/519,476, filed Nov. 12, 2003; and to U.S. Provisional PatentApplication Ser. No. 60/524,340, filed Nov. 20, 2003; and to U.S.Provisional Patent Application Ser. No. 60/532,591, filed Dec. 23, 2003.The entirety of all Provisional Applications listed above areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to phosphonate containing compounds withantiviral activity.

BACKGROUND OF THE INVENTION

Improving the delivery of drugs and other agents to target cells andtissues has been the focus of considerable research for many years.Though many attempts have been made to develop effective methods forimporting biologically active molecules into cells, both in vivo and invitro, none has proved to be entirely satisfactory. Optimizing theassociation of the inhibitory drug with its intracellular target, whileminimizing intercellular redistribution of the drug, e.g., toneighboring cells, is often difficult or inefficient.

Most agents currently administered to a patient parenterally are nottargeted, resulting in systemic delivery of the agent to cells andtissues of the body where it is unnecessary, and often undesirable. Thismay result in adverse drug side effects, and often limits the dose of adrug (e.g., glucocorticoids and other anti-inflammatory drugs) that canbe administered. By comparison, although oral administration of drugs isgenerally recognized as a convenient and economical method ofadministration, oral administration can result in either (a) uptake ofthe drug through the cellular and tissue barriers, e.g., blood/brain,epithelial, cell membrane, resulting in undesirable systemicdistribution, or (b) temporary residence of the drug within thegastrointestinal tract. Accordingly, a major goal has been to developmethods for specifically targeting agents to cells and tissues. Benefitsof such treatment includes avoiding the general physiological effects ofinappropriate delivery of such agents to other cells and tissues, suchas uninfected cells.

Thus, there is a need for therapeutic antiviral agents with improvedpharmacological properties, e.g., drugs having improved antiviralactivity and pharmacokinetic properties, including improved oralbioavailability, greater potency and extended effective half-life invivo.

New antiviral compounds should have fewer side effects, less complicateddosing schedules, and be orally active. In particular, there is a needfor a less onerous dosage regimen, such as one pill, once per day.

Assay methods capable of determining the presence, absence or amounts ofviral inhibition are of practical utility in the search for antiviral aswell as for diagnosing the presence of conditions associated infection.

SUMMARY OF THE INVENTION

Intracellular targeting may be achieved by methods and compositions thatallow accumulation or retention of biologically active agents insidecells. The present invention provides novel phosphonate analogs ofantiviral compounds. These analogs possess all the utilities of theparent compounds and optionally provide cellular accumulation as setforth below.

In one aspect, the present invention provides novel compounds withactivity against infectious viruses. The compounds of the invention mayinhibit viral RNA polymerases such as, but not limited to hepatitis B,hepatitis C, Polio, Coxsackie A and B, Rhino, Echo, small pox, Ebola,and West Nile virus polymerases. The compounds of the invention mayinhibit retroviral RNA dependent RNA polymerases or reversetranscriptases and thus inhibit the replication of the virus. Thecompounds of the invention may be useful for treating human patientsinfected with a human retrovirus, such as hepatitis C.

The present invention relates generally to the accumulation or retentionof therapeutic compounds inside cells. More particularly, the inventionrelates to attaining high concentrations of active metabolite moleculesin virally infected cells (e.g. cells infected with HCV or HIV). Sucheffective targeting may be applicable to a variety of therapeuticformulations and procedures.

Accordingly, in one embodiment the invention provides a conjugatecomprising an antiviral compound linked to one or more phosphonategroups; or a pharmaceutically acceptable salt or solvate thereof.

In another embodiment, the invention provides a compound of any one offormulae 501-561:

that is substituted with one or more groups A⁰,wherein:

A⁰ is A¹, A² or W³ with the proviso that the conjugate includes at leastone A¹;

A¹ is:

A² is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;and when Y² joins two phosphorous atoms Y² can also be C(R²)(R²);

R^(x) is independently H, R¹, R², W³, a protecting group, or theformula:

wherein:

R^(y) is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R² is independently H, R¹, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is —R^(x), —N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), —N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(Y¹)W⁵, —SO_(M2)R⁵, or —SO_(M2)W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A³ groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

X¹⁴⁹ is thymine, adenine, uracil, a 5-halouracil, a 5-alkyluracil,guanine, cytosine, a 5-halo cytosine, 5-alkyl cytosine, or2,6-diaminopurine;

X₁₅₀ is OH, Cl, NH₂, H, Me, or MeO;

X₁₅₁ is H, NH₂, or NH-alkyl;

X₁₅₂ and X₁₅₃ are independently H, alkyl, or cyclopropyl;

X₁₅₄ is a halo;

X₁₅₅ is alkoxy, aryloxy, halo-substituted alkoxy, alkenyloxy, orarylalkoxy;

X₁₅₆ is alkyl; and

X¹⁵⁷ is thymine, adenine, guanine, cytosine, uracil, inosine, ordiaminopurine.

In another embodiment the invention provides a conjugate which has theformula:

[DRUG]-(A⁰)_(nn)

wherein:

DRUG is a compound of any one of formulae 501-561;

nn is 1, 2, or 3;

A⁰ is A¹, A² or W³ with the proviso that the conjugate includes at leastone A¹;

A¹ is:

A² is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;and when Y² joins two phosphorous atoms Y² can also be C(R²)(R²);

R^(x) is independently H, R¹, R², W³, a protecting group, or theformula:

wherein:

R¹ is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R is independently H, R¹, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is R^(x), N(R^(x))(R^(x)), SR^(x), —S(O)R^(x), —S(O)₂R^(x),S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x), —SC(Y)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, C(Y¹)W⁵, SO_(M2)R⁵, or —SO_(M2)W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A³ groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

X¹⁴⁹ is thymine, adenine, uracil, a 5-halouracil, a 5-alkyluracil,guanine, cytosine, a 5-halo cytosine, 5-alkyl cytosine, or2,6-diaminopurine;

X₁₅₀ is OH, Cl, NH₂, H, Me, or MeO;

X₁₅₁ is H, NH₂, or NH-alkyl;

X₁₅₂ and X₁₅₃ are independently H, alkyl, or cyclopropyl;

X₁₅₄ is a halo;

X₁₅₅ is alkoxy, aryloxy, halo-substituted alkoxy, alkenyloxy, orarylalkoxy;

X₁₅₆ is alkyl; and

X¹⁵⁷ is thymine, adenine, guanine, cytosine, uracil, inosine, ordiaminopurine.

In another embodiment, the invention provides a conjugate of any one offormulae 1-108:

wherein:

A⁰ is A¹;

A¹ is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;and when Y² joins two phosphorous atoms Y² can also be C²R²)(R²);

R^(x) is independently H, R², W³, a protecting group, or the formula:

R^(y) is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R² is independently H, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is R^(x), —N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

R^(5a) is independently alkylene of 1 to 18 carbon atoms, alkenylene of2 to 18 carbon atoms, or alkynylene of 2-18 carbon atoms any one ofwhich alkylene, alkenylene or alkynylene is substituted with 0-3 R³groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(Y¹)W⁵, —SO₂R⁵, or —SO₂W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A³ groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

X⁵¹ is H, α-Br, or β-Br;

X⁵² is C₁-C₆ alkyl or C₇-C₁₀ arylalkyl group;

X⁵³ is H, alkyl or substituted alkyl;

X⁵⁴ is CH or N;

X⁵⁵ is thymine, adenine, uracil, a 5-halouracil, a 5-alkyluracil,guanine, cytosine, a 5-halo cytosine, a 5-alkyl cytosine, or2,6-diaminopurine;

X⁵⁶ is H, Me, Et, or i-Pr;

X⁵⁷ is H or F;

X⁵⁸ is OH, Cl, NH₂, H, Me, or MeO;

X⁵⁹ is H or NH₂;

X⁶⁰ is OH, Cl, NH₂, or H;

X⁶¹ is H, NH₂, or NH-alkyl;

X⁶² and X⁶³ are independently H, alkyl, or cyclopropyl;

X⁶⁴ is H, N₃, NH₂, or NHAc;

X⁶⁵ is a halo;

X⁶⁶ is alkoxy, aryloxy, halo-substituted alkoxy, alkenyloxy, arylalkoxy;

X⁶⁷ is O or NH;

X⁶⁸ is H, acetate, benzyl, benzyloxycarbonyl, or an amino protectinggroup;

X⁶⁹ is H or alkyl;

X⁷⁰ is H; alkyl; alkyl substituted with cycloalkyl containing three toabout six carbon atoms that is optionally substituted with one or morealkyl; alkenyl; alkenyl substituted with cycloalkyl containing three toabout six carbon atoms that is optionally substituted with one or morealkyl; hydroxyl-substituted alkyl; alkoxy-substituted alkyl;acyloxy-substituted alkyl; aryl; substituted aryl; arylalkyl; or(substituted aryl)alkyl;

X⁷¹ and X⁷² are each independently hydrogen, alkyl, phenyl, orsubstituted phenyl;

X⁷³ is alkoxy, substituted alkyl, alkylamido amino, monoalkylamino,dialkylamino, azido, chloro, hydroxy, 1-morpholino, 1-pyrrolidino, andalkylthio;

X⁷⁴ is aryl, substituted aryl, heteroaryl, or substituted heteroaryl;

X⁷⁵ and X⁷⁶ attached to nitrogen or carbon in X⁷⁴ wherein X⁷⁵ is H,halo, nitro, C₁-C₆alkyl, C₁-C₆alkoxy, (fluoro-substituted)C₁-C₆alkyl,(fluoro-substituted)C₁-C₆alkoxy, C₂-C₈alkoxyalkyl,(fluoro-substituted)C₂-C₈alkoxyalkyl, N(R^(a))(R^(b)),(CH₂)₁₋₃N(R^(a))(R^(b)), (CH₂)₀₋₃R^(c), or O(CH₂)₀₋₃R^(c); and X⁷⁶ is H,halo, nitro, C₁₋₆alkyl, C₁₋₆alkoxy, (fluoro-substituted)C₁₋₆ alkyl,(fluoro-substituted)C₁₋₆ alkoxy, C₂₋₈alkoxyalkyl,(fluoro-substituted)C₂₋₈alkoxyalkyl, N(R^(a))(R^(b)),(CH₂)₁₋₃N(R^(a))(R^(b)), (CH₂)₀₋₃R^(c), O(CH₂)₀₋₃R^(c)C, (CH₂)₀₋₃R^(d),O(CH₂)₀₋₃ R^(d), C(═O)CH₂C(═O)R^(e), or R^(f); R^(a) and R^(b) are eachindependently H, C₁-C₆ alkyl, or (fluoro-substituted)C₁-C₆ alkyl; R^(c)is aryl, or substituted aryl; R^(d) is heterocycle, or substitutedheterocycle; R^(e) is heteroaryl or substituted heteroaryl; R^(f) isX¹²⁰—NH(CH₂)₁₋₃X¹², wherein X¹²⁰ is a 5- or 6-membered monocyclicheterocycle which is saturated or unsaturated and which contains carbonatoms and from 1 to 3 nitrogen atoms and which is unsubstituted orsubstituted with one or more substituents selected from halo, cyano, OH,(CH₂)₁₋₄OH, oxo, N(R^(a))(R^(b)), C₁-C₆ alkyl, fluorinated C₁-C₆ alkyl,C₁-C₆ alkoxy, fluorinated C₁-C₆ alkoxy, (CH₂)₀₋₄CO₂R^(a),(CH₂)₀₋₄C(═O)N(R^(a))(R^(b)), (CH₂)₀₋₄SO₂R^(a), (CH₂)₁₋₄N(R^(a))(R^(b)),(CH₂)₀₋₄N(R^(a))C(═O)R^(b), (CH₂)₀₋₄SO₂N(R^(a))(R^(b)),(CH₂)₁₋₄N(R^(a))SO₂R^(b), C₂-C₈ alkoxyalkyl, and(fluoro-substituted)C₂-C₈ alkoxyalkyl; and X¹²¹ is pyrrolidinyl,piperidinyl, piperazinyl, or morpholinyl, which is unsubstituted orsubstituted with one or more substituents selected from halo, cyano, OH,(CH₂)₁₋₄OH, oxo, N(R^(a))(R^(b)), C₁-C₆ alkyl, fluorinated C₁-C₆ alkyl,C₁-C₆ alkoxy, fluorinated C₁-C₆ alkoxy, (CH₂)₀₋₄CO₂R^(a),(CH₂)₀₋₄C(═O)N(R^(a))(R^(b)), (CH₂)₀₋₄SO₂R^(a), (CH₂)₁₋₄N(R^(a))(R^(b)),(CH₂)₀₋₄N(R^(a))C(═O)R^(b), (CH₂)₀₋₄SO₂N(R^(a))(R^(b)),(CH₂)₁₋₄N(R^(a))SO₂R^(b), C₂-C₈ alkoxyalkyl, and(fluoro-substituted)C₂-C₈ alkoxyalkyl;

X⁷⁷ is H or C₁₋₆ alkyl;

X⁷⁸ is OH, protected hydroxyl, or N(R^(a))(R^(b));

X⁷⁹ is a attached to nitrogen or carbon in X⁷⁴; and X⁷⁹ is H, halo,nitro, oxo, C₁₋₆alkyl, C₃₋₇cycloalkyl, C₃₋₇cycloalkoxy, C₁₋₆alkoxy,(fluoro-substituted)C₁₋₆ alkyl, (fluoro-substituted)C₁₋₆ alkoxy, C₂₋₈alkoxyalkyl, (fluoro-substituted)C₂₋₈ alkoxyalkyl, N(R^(a))(R^(b)),(CH₂)₁₋₄N(R^(a))(R^(b)), C(═O)N(R^(a))(R^(b)), (CH₂)₁₋₄C(═O)N(R^(a))(R^(b)), N(R^(a))C(═O)R^(b), (CH₂)₁₋₄N(R^(a))C(═O)R^(b),SO₂R^(a) (CH₂)₁₋₄ SO₂R^(a), SO₂N(R^(a))(R^(b)),(CH₂)₁₋₄SO₂N(R^(a))(R^(b)), (CH₂)₁₋₄N(R^(a))SO₂R^(b), (CH₂)₀₋₃R^(c), or(CH₂)₀₋₃R^(g);

R^(g) is a 5- or 6-membered monocyclic heterocycle which is saturated orunsaturated and which contains one or more carbon atoms and from 1 to 4nitrogen atoms, the heterocycle being unsubstituted or substituted withone or more substituents selected from halo, cyano, OH, (CH₂)₁₋₄OH, oxo,N(R^(a))(R^(b)), C₁-C₆alkyl, (fluoro-substituted)C₁-C₆alkyl,C₁-C₆alkoxy, (fluoro-substituted)C₁-C₆ alkoxy, (CH₂)₀₋₄CO₂R^(a),(CH₂)₀₋₄C(═O)N(R^(a))(R^(b)), (CH₂)₀₋₄SO₂R^(a), (CH₂)₁₋₄N(R^(a))(R^(b)),(CH₂)₀₋₄N(R^(a))C(═O)R^(b), (CH₂)₀₋₄SO₂N(R^(a))(R^(b)),(CH₂)₁₋₄N(R^(a))SO₂R^(b), C₂-C₈alkoxyalkyl,(fluoro-substituted)C₂-C₈alkoxyalkyl, phenyl and benzyl;

X⁸⁰ is (i) a 5- or 6-membered heteroaromatic ring containing from 1 to 4nitrogen atoms, 0 to 2 sulfur atoms, and at least 1 carbon atom, or (ii)an 8- to 10-membered fused bicyclic heterocycle containing from 1 to 4nitrogen atoms, 0 to 2 sulfur atoms, and carbon atoms, wherein the ringof the heterocycle attached to the central dione moiety is a 5- or 6membered heteroaromatic ring containing at least one nitrogen or sulfuratom and the other ring of the heterocycle is a saturated or unsaturatedring; wherein X⁸⁰ is attached to the central propenone moiety via acarbon atom and at least one nitrogen or sulfur atom in X⁸⁰ is adjacentto the point of attachment;

X⁸¹ is attached to nitrogen or carbon in X⁸⁰, and is independentlyselected from H, halo, OH, (CH₂)₁₋₄OH, C₁-C₆ alkyl, C₁-C₆ alkoxy,(fluoro-substituted)C₁-C₆ alkyl, (fluoro-substituted)C₁-C₆ alkoxy, C₁-C₈alkoxyalkyl, (fluoro-substituted)C₁-C₈ alkoxyalkyl, N(R^(a))(R^(b)),(CH₂)₁₋₄N(R^(a))(R^(b)), C(═O)N(R^(a))(R^(b)),(CH₂)₁₋₄C(═O)N(R^(a))(R^(b)), N(R^(a))C(═O)R^(b),(CH₂)₁₋₄N(R^(a))C(═O)R^(b), SO₂R^(a), (CH₂)₁₋₄SO₂R^(a),SO₂N(R^(a))(R^(b)), (CH₂)₁₋₄SO₂N(R^(a))(R^(b)),(CH₂)₁₋₄N(R^(a))SO₂R^(b), and (CH₂)₀₋₃R^(b);

X⁸² is OH, F, or cyano;

X⁸³ is N or CH;

X⁸⁴ is cis H or trans H;

X⁸⁵ is C₈-C₁₆ alkyl which can optionally contain one to five oxygenatoms in the chain;

X⁸⁶ is H, methyl, hydroxymethyl, or fluoromethyl;

X⁸⁷ and X⁸⁸ are each independently H or C₁₋₄ alkyl, which alkyl isoptionally substituted with OH, amino, C₁₋₄ alkoxy, C₁₋₄ alkylthio, orone to three halogen atoms;

X⁸⁹ is —O— or —S(O)_(n)—, where n is 0, 1, or 2;

X⁹⁰ is H, methyl, hydroxymethyl, or fluoromethyl;

X⁹¹ is H hydroxy, alkyl, azido, cyano, alkenyl, alkynyl, bromovinyl,—C(O)O(alkyl), —O(acyl), alkoxy, alkenyloxy, chloro, bromo, fluoro,iodo, NO₂, NH₂, —NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂,—N(acyl)₂;

X⁹² is H, C₂₋₄alkenyl, C₂₋₄alkynyl, or C₁₋₄ alkyl optionally substitutedwith amino, hydroxy, or 1 to 3 fluorine atoms;

one of X⁹³ and X⁹⁴ is hydroxy or C₁₋₄ alkoxy and the other of X⁹³ andX⁹⁴ is selected from the group consisting of H; hydroxy; halo; C₁₋₄alkyl optionally substituted with 1 to 3 fluorine atoms; C₁₋₁₀ alkoxy,optionally substituted with C₁₋₃ alkoxy or 1 to 3 fluorine atoms; C₂₋₆alkenyloxy; C₁₋₄alkylthio; C₁₋₈ alkylcarbonyloxy; aryloxycarbonyl;azido; amino; C₁₋₄ alkylamino; and di(C₁₋₄ alkyl)amino; or

X⁹³ is H, C₂₋₄ alkenyl, C₂₋₄ alkynyl, or C₁₋₄ alkyl optionallysubstituted with amino, hydroxy, or 1 to 3 fluorine atoms, and one ofX⁹² and X⁹⁴ is hydroxy or C₁₋₄alkoxy and the other of X⁹² and X⁹⁴ isselected from the group consisting of H; hydroxy; halo; C₁₋₄ alkyloptionally substituted with 1 to 3 fluorine atoms; C₁₋₁₀ alkoxy,optionally substituted with C₁₋₃ alkoxy or 1 to 3 fluorine atoms; C₂₋₆alkenyloxy; C₁₋₄alkylthio; C₁₋₈ alkylcarbonyloxy; aryloxycarbonyl;azido; amino; C₁₋₄ alkylamino; and di(C₁₋₄ alkyl)amino; or

X⁹² and X⁹³ together with the carbon atom to which they are attachedform a 3- to 6 membered saturated monocyclic ring system optionallycontaining a heteroatom selected from O, S, and NCO₄ alkyl;

X⁹⁵ is H, OH, SH, NH₂, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino,C₃₋₆cycloalkylamino, halo, C₁₋₄alkyl, C₁₋₄ alkoxy, or CF₃; or X⁹² andX⁹⁵ can optionally together be a bond linking the two carbons to whichthey are attached;

X⁹⁶ is H, methyl, hydroxymethyl, or fluoromethyl;

X⁹⁷ is selected from the group consisting of

U, G, and J are each independently CH or N;

D is N, CH, C—CN, C—NO₂, C—C₁₋₃ alkyl, C—NHCONH₂, C—CONT₁₁T₁₁, C—CSNT₁₁T₁₁, C—COOT₁₁, C—C(═NH)NH₂, C-hydroxy, C—C₁₋₃ alkoxy, C-amino, C—C₁₋₄alkylamino, C-di(C₁₋₄ alkyl)amino, C-halogen, C-(1,3-oxazol-2-yl),C-(1,3 thiazol-2-yl), or C-(imidazol-2-yl); wherein alkyl isunsubstituted or substituted with one to three groups independentlyselected from halogen, amino, hydroxy, carboxy, and C₁₋₃ alkoxy;

E is N or CT₅;

W^(a) is O or S;

T₁ is H, C₂₋₄alkenyl, C₂₋₄alkynyl, or C₁₋₄alkyl optionally substitutedwith amino, hydroxy, or 1 to 3 fluorine atoms and one of T₂ and T₃ ishydroxy or C₁₋₄ alkoxy and the other of T₂ and T₃ is selected from thegroup consisting of H; hydroxy; halo; C₁₋₄ alkyl optionally substitutedwith 1 to 3 fluorine atoms; C₁₋₁₀ alkoxy, optionally substituted withC₁₋₃ alkoxy or 1 to 3 fluorine atoms; C₂₋₆ alkenyloxy; C₁₋₄alkylthio;C₁₋₈ alkylcarbonyloxy; aryloxycarbonyl; azido; amino; C₁₋₄ alkylamino;and di(C₁₋₄ alkyl)amino; or

T₂ is H, C₂₋₄alkenyl, C₂₋₄alkynyl, or C₁₋₄alkyl optionally substitutedwith amino, hydroxy, or 1 to 3 fluorine atoms and one of T₁ and T₃ ishydroxy or C₁₋₄alkoxy and the other of T₁ and T₃ is selected from thegroup consisting of H; hydroxy; halo; C₁₋₄ alkyl optionally substitutedwith 1 to 3 fluorine atoms; C₁₋₁₀ alkoxy, optionally substituted withC₁₋₃ alkoxy or 1 to 3 fluorine atoms; C₂₋₆ alkenyloxy; C₁₋₄alkylthio;C₁₋₈ alkylcarbonyloxy; aryloxycarbonyl; azido; amino; C₁₋₄ alkylamino;and di(C₁₋₄ alkyl)amino; or

T₁ and T₂ together with the carbon atom to which they are attached forma 3- to 6 membered saturated monocyclic ring system optionallycontaining a heteroatom selected from O, S, and NC₀₋₄ alkyl;

T₄ and T₆ are each independently H, OH, SH, NH₂, C₁₋₄ alkylamino,di(C₁₋₄ alkyl)amino, C₃₋₆cycloalkylamino, halo, C₁₋₄ alkyl, C₁₋₄ alkoxy,or CF₃;

T₅ is H, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₄alkylamino, CF₃, orhalogen; T₁₄ is H, CF₃, C₁₋₄ alkyl, amino, C₁₋₄alkylamino,C₃₋₆cycloalkylamino, or di(C₁₋₄alkyl)amino;

T₇ is H, amino, C₁₋₄alkylamino, C₃₋₆ cycloalkylamino, ordi(C₁₋₄alkyl)amino;

each T₁₁ is independently H or C₁₋₆ alkyl;

T₈ is H, halo, CN, carboxy, C₁₋₄ alkyloxycarbonyl, N₃, amino, C₁₋₄alkylamino, di(C₁₋₄ alkyl)amino, hydroxy, C₁₋₆ alkoxy, C₁₋₆ alkylthio,C₁₋₆ alkylsulfonyl, or (C₁₋₄ alkyl)₀₋₂ aminomethyl;

X⁹⁸ is methoxy, ethoxy, vinyl, ethyl, methyl, cyclopropyl,N-methylamino, or N-formylamino;

X⁹⁹ is methyl, chloro, or trifluoromethyl;

X¹⁰⁰ is H, methyl, ethyl, cyclopropyl, vinyl, or trifluoromethyl;

X¹⁰¹ is H, methyl, ethyl, cyclopropyl, chloro, vinyl, allyl,3-methyl-1-buten-yl;

X¹⁰² is thymine, adenine, guanine, cytosine, uracil, inosine, ordiaminopurine;

X¹⁰³ is OH, OR, NR₂, CN, NO₂, F, Cl, Br, or I;

X¹⁰⁴ is adenine, guanine, cytosine, uracil, thymine, 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine,nebularine, nitropyrrole, nitroindole, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, 06-methylguanine, N⁶-methyladenine,O⁴-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole,or pyrazolo[3,4-d]pyrimidine;

X¹⁰⁵ is selected from O, C(R^(y))₂, OC(R^(y))₂, NR and S;

X¹⁰⁶ is selected from O, C(R^(y))₂, C═C(R^(y))₂, NR and S;

X¹⁰⁷ is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-D]pyrimidine;

X¹⁰⁸ is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I;

X¹⁰⁹ is selected from H, C₁-C₈alkyl, substituted C₁-C₈alkyl,C₁-C₈alkenyl, substituted C₁-C₈alkenyl, C₁-C₈alkynyl, and substitutedC₁-C₈alkynyl,

X¹¹⁰ is independently O, CR₂, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S,S—S, S(O), or S(O)₂;

X¹¹¹ is adenine, guanine, cytosine, uracil, thymine, 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine,nebularine, nitropyrrole, nitroindole, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O⁶-methylguanine, N⁶-methyladenine,O⁴-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole,substituted triazole, or pyrazolo[3,4-D]pyrimidine;

X¹¹² is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I;

X¹¹³ is F;

X¹¹⁴ is independently H, F, Cl, Br, I, OH, R, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₈alkyl, carboxy, sulfate, sulfamate,sulfonate, 5-7 membered ring sultam, C₁-C₈ alkylsulfonate,4-dialkylaminopyridinium, hydroxyl-substituted C₁-C₈alkyl,C₁-C₈alkylthiol, alkylsulfonyl, arylsulfonyl, arylsulfinyl (—SOAr),arylthio, —SO₂NR₂, —SOR, —C(═O)OR, —C(═O)NR₂, 5-7 membered ring lactam,5-7 membered ring lactone, cyano, azido, nitro, C₁-C₈alkoxy, substitutedC₁-C₈alkyl, C₁-C₈alkenyl, substituted C₁-C₈alkenyl, C₁-C₈alkynyl,substituted C₁-C₈ alkynyl, aryl, substituted aryl, heterocycle,substituted heterocycle, polyethyleneoxy, a protecting group, or W³; orwhen taken together, two Rys form a carbocyclic ring of 3 to 7 carbonatoms;

X¹¹⁵ is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I; and

X¹¹⁶ is selected from H, C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, and C₁-C₈ substitutedalkynyl.

The invention provides a pharmaceutical composition comprising aneffective amount of a conjugate of the invention, or a pharmaceuticallyacceptable salt or solvate thereof, and a pharmaceutically acceptableexcipient.

This invention pertains to a method of increasing cellular accumulationand retention of an anti-viral compound comprising linking the compoundto one or more phosphonate groups.

The invention also provides a method of inhibiting a viral infection inan animal (e.g. a mammal), comprising administering an effective amounta conjugate of the invention to the animal.

The invention also provides a compound of the invention for use inmedical therapy (preferably for use in treating a viral infection in ananimal), as well as the use of a compound of the invention for themanufacture of a medicament useful for the treatment of a viralinfection in an animal (e.g. a mammal).

The invention also provides processes and novel intermediates disclosedherein which are useful for preparing compounds of the invention. Someof the compounds of the invention are useful to prepare other compoundsof the invention.

In another embodiment the invention provides a method for inhibiting aviral infection in a sample comprising treating a sample suspected ofcontaining a virus, with a compound or composition of the invention.

DETAILED DESCRIPTION OF EXEMPLARY CLAIMS

Reference will now be made in detail to certain claims of the invention,examples of which are illustrated in the accompanying structures andformulas. While the invention will be described in conjunction with theenumerated claims, it will be understood that they are not intended tolimit the invention to those claims. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

When tradenames are used herein, applicants intend to independentlyinclude the tradename product and the active pharmaceuticalingredient(s) of the tradename product.

“Bioavailability” is the degree to which the pharmaceutically activeagent becomes available to the target tissue after the agent'sintroduction into the body. Enhancement of the bioavailability of apharmaceutically active agent can provide a more efficient and effectivetreatment for patients because, for a given dose, more of thepharmaceutically active agent will be available at the targeted tissuesites.

The terms “phosphonate” and “phosphonate group” include functionalgroups or moieties within a molecule that comprises a phosphorous thatis 1) single-bonded to a carbon, 2) double-bonded to a heteroatom, 3)single-bonded to a heteroatom, and 4) single-bonded to anotherheteroatom, wherein each heteroatom can be the same or different. Theterms “phosphonate” and “phosphonate group” also include functionalgroups or moieties that comprise a phosphorous in the same oxidationstate as the phosphorous described above, as well as functional groupsor moieties that comprise a prodrug moiety that can separate from acompound so that the compound retains a phosphorous having thecharacteristics described above. For example, the terms “phosphonate”and “phosphonate group” include phosphonic acid, phosphonic monoester,phosphonic diester, phosphonamidate, and phosphonthioate functionalgroups. In one specific embodiment of the invention, the terms“phosphonate” and “phosphonate group” include functional groups ormoieties within a molecule that comprises a phosphorous that is 1)single-bonded to a carbon, 2) double-bonded to an oxygen, 3)single-bonded to an oxygen, and 4) single-bonded to another oxygen, aswell as functional groups or moieties that comprise a prodrug moietythat can separate from a compound so that the compound retains aphosphorous having such characteristics. In another specific embodimentof the invention, the terms “phosphonate” and “phosphonate group”include functional groups or moieties within a molecule that comprises aphosphorous that is 1) single-bonded to a carbon, 2) double-bonded to anoxygen, 3) single-bonded to an oxygen or nitrogen, and 4) single-bondedto another oxygen or nitrogen, as well as functional groups or moietiesthat comprise a prodrug moiety that can separate from a compound so thatthe compound retains a phosphorous having such characteristics.

The term “prodrug” as used herein refers to any compound that whenadministered to a biological system generates the drug substance, i.e.active ingredient, as a result of spontaneous chemical reaction(s),enzyme catalyzed chemical reaction(s), photolysis, and/or metabolicchemical reaction(s). A prodrug is thus a covalently modified analog orlatent form of a therapeutically-active compound.

“Prodrug moiety” refers to a labile functional group which separatesfrom the active inhibitory compound during metabolism, systemically,inside a cell, by hydrolysis, enzymatic cleavage, or by some otherprocess (Bundgaard, Hans, “Design and Application of Prodrugs” in ATextbook of Drug Design and Development (1991), P. Krogsgaard-Larsen andH. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191). Enzymeswhich are capable of an enzymatic activation mechanism with thephosphonate prodrug compounds of the invention include, but are notlimited to, amidases, esterases, microbial enzymes, phospholipases,cholinesterases, and phosphases. Prodrug moieties can serve to enhancesolubility, absorption and lipophilicity to optimize drug delivery,bioavailability and efficacy. A prodrug moiety may include an activemetabolite or drug itself.

Exemplary prodrug moieties include the hydrolytically sensitive orlabile acyloxymethyl esters —CH₂C(═O)R⁹ and acyloxymethyl carbonates—CH₂C(═O)OR⁹ where R⁹ is C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₆-C₂₀aryl or C₆-C₂₀ substituted aryl. The acyloxyalkyl ester was first usedas a prodrug strategy for carboxylic acids and then applied tophosphates and phosphonates by Farquhar et al. (1983) J. Pharm. Sci. 72:324; also U.S. Pat. Nos. 4,816,570, 4,968,788, 5,663,159 and 5,792,756.Subsequently, the acyloxyalkyl ester was used to deliver phosphonicacids across cell membranes and to enhance oral bioavailability. A closevariant of the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl ester(carbonate), may also enhance oral bioavailability as a prodrug moietyin the compounds of the combinations of the invention. An exemplaryacyloxymethyl ester is pivaloyloxymethoxy, (POM) —CH₂C(═O)C(CH₃)₃. Anexemplary acyloxymethyl carbonate prodrug moiety ispivaloyloxymethylcarbonate (POC)—CH₂C(═O)OC(CH₃)₃.

The phosphonate group may be a phosphonate prodrug moiety. The prodrugmoiety may be sensitive to hydrolysis, such as, but not limited to apivaloyloxymethyl carbonate (POC) or POM group. Alternatively, theprodrug moiety may be sensitive to enzymatic potentiated cleavage, suchas a lactate ester or a phosphonamidate-ester group.

Aryl esters of phosphorus groups, especially phenyl esters, are reportedto enhance oral bioavailability (De Lombaert et al. (1994) J. Med. Chem.37: 498). Phenyl esters containing a carboxylic ester ortho to thephosphate have also been described (Khamnei and Torrence, (1996) J. Med.Chem. 39:4109-4115). Benzyl esters are reported to generate the parentphosphonic acid. In some cases, substituents at the ortho- orpara-position may accelerate the hydrolysis. Benzyl analogs with anacylated phenol or an alkylated phenol may generate the phenoliccompound through the action of enzymes, e.g., esterases, oxidases, etc.,which in turn undergoes cleavage at the benzylic C—O bond to generatethe phosphoric acid and the quinone methide intermediate. Examples ofthis class of prodrugs are described by Mitchell et al. (1992) J. Chem.Soc. Perkin Trans. II 2345; Glazier WO 91/19721. Still other benzylicprodrugs have been described containing a carboxylic ester-containinggroup attached to the benzylic methylene (Glazier WO 91/19721).Thio-containing prodrugs are reported to be useful for the intracellulardelivery of phosphonate drugs. These proesters contain an ethylthiogroup in which the thiol group is either esterified with an acyl groupor combined with another thiol group to form a disulfide.Deesterification or reduction of the disulfide generates the free thiointermediate which subsequently breaks down to the phosphoric acid andepisulfide (Puech et al. (1993) Antiviral Res., 22: 155-174; Benzaria etal. (1996) J. Med. Chem. 39: 4958). Cyclic phosphonate esters have alsobeen described as prodrugs of phosphorus-containing compounds (Erion etal., U.S. Pat. No. 6,312,662).

“Protecting group” refers to a moiety of a compound that masks or altersthe properties of a functional group or the properties of the compoundas a whole. Chemical protecting groups and strategies forprotection/deprotection are well known in the art. See e.g., ProtectiveGroups in Organic Chemistry, Theodora W. Greene, John Wiley & Sons,Inc., New York, 1991. Protecting groups are often utilized to mask thereactivity of certain functional groups, to assist in the efficiency ofdesired chemical reactions, e.g., making and breaking chemical bonds inan ordered and planned fashion. Protection of functional groups of acompound alters other physical properties besides the reactivity of theprotected functional group, such as the polarity, lipophilicity(hydrophobicity), and other properties which can be measured by commonanalytical tools. Chemically protected intermediates may themselves bebiologically active or inactive.

Protected compounds may also exhibit altered, and in some cases,optimized properties in vitro and in vivo, such as passage throughcellular membranes and resistance to enzymatic degradation orsequestration. In this role, protected compounds with intendedtherapeutic effects may be referred to as prodrugs. Another function ofa protecting group is to convert the parental drug into a prodrug,whereby the parental drug is released upon conversion of the prodrug invivo. Because active prodrugs may be absorbed more effectively than theparental drug, prodrugs may possess greater potency in vivo than theparental drug. Protecting groups are removed either in vitro, in theinstance of chemical intermediates, or in vivo, in the case of prodrugs.With chemical intermediates, it is not particularly important that theresulting products after deprotection, e.g., alcohols, bephysiologically acceptable, although in general it is more desirable ifthe products are pharmacologically innocuous.

Any reference to any of the compounds of the invention also includes areference to a physiologically acceptable salt thereof. Examples ofphysiologically acceptable salts of the compounds of the inventioninclude salts derived from an appropriate base, such as an alkali metal(for example, sodium), an alkaline earth (for example, magnesium),ammonium and NX₄ ⁺ (wherein X is C₁-C₄ alkyl). Physiologicallyacceptable salts of an hydrogen atom or an amino group include salts oforganic carboxylic acids such as acetic, benzoic, lactic, fumaric,tartaric, maleic, malonic, malic, isethionic, lactobionic and succinicacids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic,benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, suchas hydrochloric, sulfuric, phosphoric and sulfamic acids.Physiologically acceptable salts of a compound of an hydroxy groupinclude the anion of said compound in combination with a suitable cationsuch as Na⁺ and NX₄ ⁺ (wherein X is independently selected from H or aC₁-C₄ alkyl group).

For therapeutic use, salts of active ingredients of the compounds of theinvention will be physiologically acceptable, i.e. they will be saltsderived from a physiologically acceptable acid or base. However, saltsof acids or bases which are not physiologically acceptable may also finduse, for example, in the preparation or purification of aphysiologically acceptable compound. All salts, whether or not derivedform a physiologically acceptable acid or base, are within the scope ofthe present invention.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto, ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂)—

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to,acetylenic (—C═CH) and propargyl (—CH₂C═CH),

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to, methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to, 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to, acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. The arylalkyl group comprises 6to 20 carbon atoms, e.g., the alkyl moiety, including alkanyl, alkenylor alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and thearyl moiety is 5 to 14 carbon atoms.

“Substituted alkyl”, “substituted aryl”, and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a non-hydrogensubstituent. Typical substituents include, but are not limited to, —X,—R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O,—NCS, —NO, —NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NRR —S(═O)₂O⁻,—S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)O₂RR,—P(═O)O₂RR—P(═O)(O⁻)₂, —P(═O)(OH)₂, —C(═O)R, —C(═O)X, —C(S)R, —C(O)OR,—C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR, —C(NR)NRR, whereeach X is independently a halogen: F, Cl, Br, or I; and each R isindependently —H, alkyl, aryl, heterocycle, protecting group or prodrugmoiety. Alkylene, alkenylene, and alkynylene groups may also besimilarly substituted.

“Heterocycle” as used herein includes by way of example and notlimitation these heterocycles described in Paquette, Leo A.; Principlesof Modern Heterocyclic Chemistry (W. A. Benjamin, New York, 1968),particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry ofHeterocyclic Compounds, A Series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment of theinvention “heterocycle” includes a “carbocycle” as defined herein,wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replacedwith a heteroatom (e.g. O, N, or S).

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,isatinoyl, and bis-tetrahydrofuranyl:

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

“Carbocycle” refers to a saturated, unsaturated or aromatic ring having3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle,and up to about 20 carbon atoms as a polycycle. Monocyclic carbocycleshave 3 to 6 ring atoms, still more typically 5 or 6 ring atoms. Bicycliccarbocycles have 7 to 12 ring atoms, e.g., arranged as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as abicyclo [5,6] or [6,6] system. Examples of monocyclic carbocyclesinclude cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl, spiryl and naphthyl.

“Linker” or “link” refers to a chemical moiety comprising a covalentbond or a chain or group of atoms that covalently attaches a phosphonategroup to a drug. Linkers include portions of substituents A¹ and A³,which include moieties such as: repeating units of alkyloxy (e.g.,polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g.,polyethyleneamino, Jeffamine™); and diacid ester and amides includingsuccinate, succinamide, diglycolate, malonate, and caproamide.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

The term “treatment” or “treating,” to the extent it relates to adisease or condition includes preventing the disease or condition fromoccurring, inhibiting the disease or condition, eliminating the diseaseor condition, and/or relieving one or more symptoms of the disease orcondition.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and l or (+) and (−) are employed todesignate the sign of rotation of plane-polarized light by the compound,with (−) or l meaning that the compound is levorotatory. A compoundprefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

Protecting Groups

In the context of the present invention, protecting groups includeprodrug moieties and chemical protecting groups.

Protecting groups are available, commonly known and used, and areoptionally used to prevent side reactions with the protected groupduring synthetic procedures, i.e. routes or methods to prepare thecompounds of the invention. For the most part the decision as to whichgroups to protect, when to do so, and the nature of the chemicalprotecting group “PG” will be dependent upon the chemistry of thereaction to be protected against (e.g., acidic, basic, oxidative,reductive or other conditions) and the intended direction of thesynthesis. The PG groups do not need to be, and generally are not, thesame if the compound is substituted with multiple PG. In general, PGwill be used to protect functional groups such as carboxyl, hydroxyl,thio, or amino groups and to thus prevent side reactions or to otherwisefacilitate the synthetic efficiency. The order of deprotection to yieldfree, deprotected groups is dependent upon the intended direction of thesynthesis and the reaction conditions to be encountered, and may occurin any order as determined by the artisan.

Various functional groups of the compounds of the invention may beprotected. For example, protecting groups for —OH groups (whetherhydroxyl, carboxylic acid, phosphonic acid, or other functions) include“ether- or ester-forming groups”. Ether- or ester-forming groups arecapable of functioning as chemical protecting groups in the syntheticschemes set forth herein. However, some hydroxyl and thio protectinggroups are neither ether- nor ester-forming groups, as will beunderstood by those skilled in the art, and are included with amides,discussed below.

A very large number of hydroxylprotecting groups and amide-forminggroups and corresponding chemical cleavage reactions are described inProtective Groups in Organic Synthesis, Theodora W. Greene (John Wiley &Sons, Inc., New York, 1991, ISBN 0-471-62301-6) (“Greene”). See alsoKocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart,New York, 1994), which is incorporated by reference in its entiretyherein. In particular Chapter 1, Protecting Groups: An Overview, pages1-20, Chapter 2, Hydroxyl Protecting Groups, pages 21-94, Chapter 3,Diol Protecting Groups, pages 95-117, Chapter 4, Carboxyl ProtectingGroups, pages 118-154, Chapter 5, Carbonyl Protecting Groups, pages155-184. For protecting groups for carboxylic acid, phosphonic acid,phosphonate, sulfonic acid and other protecting groups for acids seeGreene as set forth below. Such groups include by way of example and notlimitation, esters, amides, hydrazides, and the like.

Ether- and Ester-forming Protecting Groups

Ester-forming groups include: (1) phosphonate ester-forming groups, suchas phosphonamidate esters, phosphorothioate esters, phosphonate esters,and phosphon-bis-amidates; (2) carboxyl ester-forming groups, and (3)sulphur ester-forming groups, such as sulphonate, sulfate, andsulfinate.

The phosphonate moieties of the compounds of the invention may or maynot be prodrug moieties, i.e. they may or may be susceptible tohydrolytic or enzymatic cleavage or modification. Certain phosphonatemoieties are stable under most or nearly all metabolic conditions. Forexample, a dialkylphosphonate, where the alkyl groups are two or morecarbons, may have appreciable stability in vivo due to a slow rate ofhydrolysis.

Within the context of phosphonate prodrug moieties, a large number ofstructurally-diverse prodrugs have been described for phosphonic acids(Freeman and Ross in Progress in Medicinal Chemistry 34: 112-147 (1997)and are included within the scope of the present invention. An exemplaryphosphonate ester-forming group is the phenyl carbocycle in substructureA₃ having the formula:

wherein R₁ may be H or C₁-C₁₂ alkyl; m1 is 1, 2, 3, 4, 5, 6, 7 or 8, andthe phenyl carbocycle is substituted with 0 to 3 R₂ groups. Where Y, isO, a lactate ester is formed, and where Y₁ is N(R₂), N(OR₂) or N(N(R₂)₂,a phosphonamidate ester results.

In its ester-forming role, a protecting group typically is bound to anyacidic group such as, by way of example and not limitation, a —CO₂H or—C(S)OH group, thereby resulting in —CO₂R^(x) where R^(x) is definedherein. Also, R^(x) for example includes the enumerated ester groups ofWO 95/07920.

Examples of protecting groups include:

C₃-C₁₂ heterocycle (described above) or aryl. These aromatic groupsoptionally are polycyclic or monocyclic. Examples include phenyl,spiryl, 2- and 3-pyrrolyl, 2- and 3-thienyl, 2- and 4-imidazolyl, 2-, 4-and 5-oxazolyl, 3- and 4-isoxazolyl, 2-, 4- and 5-thiazolyl, 3-, 4- and5-isothiazolyl, 3- and 4-pyrazolyl, 1-, 2-, 3- and 4-pyridinyl, and 1-,2-, 4- and 5-pyrimidinyl,

C₃-C₁₂ heterocycle or aryl substituted with halo, R¹, R′—O—C₁-C₁₂alkylene, C₁-C₁₂ alkoxy, CN, NO₂, OH, carboxy, carboxyester, thiol,thioester, C₁-C₁₂ haloalkyl (1-6 halogen atoms), C₂-C₁₂ alkenyl orC₂-C₁₂ alkynyl. Such groups include 2-, 3- and 4-alkoxyphenyl (C₁-C₁₂alkyl), 2-, 3- and 4-methoxyphenyl, 2-, 3- and 4-ethoxyphenyl, 2,3-,2,4-, 2,5-, 2,6-, 3,4- and 3,5-diethoxyphenyl, 2- and3-carboethoxy-4-hydroxyphenyl, 2- and 3-ethoxy-4-hydroxyphenyl, 2- and3-ethoxy-5-hydroxyphenyl, 2- and 3-ethoxy-6-hydroxyphenyl, 2-, 3- and4-O-acetylphenyl, 2-, 3- and 4-dimethylaminophenyl, 2-, 3- and4-methylmercaptophenyl, 2-, 3- and 4-halophenyl (including 2-, 3- and4-fluorophenyl and 2-, 3- and 4-chlorophenyl), 2,3-, 2,4-, 2,5-, 2,6-,3,4- and 3,5-dimethylphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and3,5-biscarboxyethylphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and3,5-dimethoxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dihalophenyl(including 2,4-difluorophenyl and 3,5-difluorophenyl), 2-, 3- and4-haloalkylphenyl (1 to 5 halogen atoms, C₁-C₁₂ alkyl including4-trifluoromethylphenyl), 2-, 3- and 4-cyanophenyl, 2-, 3- and4-nitrophenyl, 2-, 3- and 4-haloalkylbenzyl (1 to 5 halogen atoms,C₁-C₁₂ alkyl including 4-trifluoromethylbenzyl and 2-, 3- and4-trichloromethylphenyl and 2-, 3- and 4-trichloromethylphenyl),4-N-methylpiperidinyl, 3-N-methylpiperidinyl, 1-ethylpiperazinyl,benzyl, alkylsalicylphenyl (C₁-C₄ alkyl, including 2-, 3- and4-ethylsalicylphenyl), 2-, 3- and 4-acetylphenyl, 1,8-dihydroxynaphthyl(—C₁₀H₆—OH) and aryloxy ethyl [C₆-C₈ aryl (including phenoxy ethyl)],2,2′-dihydroxybiphenyl, 2-, 3- and 4-N,N-dialkylaminophenol,—C₆H₄—CH₂—N(CH₃)₂, trimethoxybenzyl, triethoxybenzyl, 2-alkyl pyridinyl(C₁₋₄ alkyl);

esters of 2-carboxyphenyl; and C₁-C₄ alkylene-C₃-C₆ aryl (includingbenzyl, —CH₂-pyrrolyl, —CH₂-thienyl, —CH₂-imidazolyl, —CH₂-oxazolyl,—CH₂-isoxazolyl, —CH₂-thiazolyl, —CH₂-isothiazolyl, —CH₂-pyrazolyl,—CH₂-pyridinyl and —CH₂-pyrimidinyl) substituted in the aryl moiety by 3to 5 halogen atoms or 1 to 2 atoms or groups selected from halogen,C₁-C₁₂ alkoxy (including methoxy and ethoxy), cyano, nitro, OH, C₁-C₁₂haloalkyl (1 to 6 halogen atoms; including —CH₂CCl₃), C₁-C₁₂ alkyl(including methyl and ethyl), C₂-C₁₂ alkenyl or C₂-C₁₂ alkynyl; alkoxyethyl [C₁-C₆ alkyl including —CH₂—CH₂—O—CH₃ (methoxy ethyl)]; alkylsubstituted by any of the groups set forth above for aryl, in particularOH or by 1 to 3 halo atoms (including —CH₃, —CH(CH₃)₂, —C(CH₃)₃,—CH₂CH₃, —(CH₂)₂CH₃, —(CH₂)₃CH₃, —(CH₂)₄CH₃, —(CH₂)₅CH₃, —CH₂CH₂F,—CH₂CH₂Cl, —CH₂CF₃, and —CH₂CCl₃);

—N-2-propylmorpholino, 2,3-dihydro-6-hydroxyindene, sesamol, catecholmonoester, —CH₂—C(O)—N(R¹)₂, —CH₂—S(O)(R¹), —CH₂—S(O)₂(R¹),—CH₂—CH(OC(O)CH₂R¹)—CH₂(OC(O)CH₂R¹), cholesteryl, enolpyruvate(HOOC—C(═CH₂)—), glycerol;

a 5 or 6 carbon monosaccharide, disaccharide or oligosaccharide (3 to 9monosaccharide residues);

triglycerides such as α-D-β-diglycerides (wherein the fatty acidscomposing glyceride lipids generally are naturally occurring saturatedor unsaturated C₆₋₂₆, C₆₋₁₈ or C₆₋₁₀ fatty acids such as linoleic,lauric, myristic, palmitic, stearic, oleic, palmitoleic, linolenic andthe like fatty acids) linked to acyl of the parental compounds hereinthrough a glyceryl oxygen of the triglyceride;

phospholipids linked to the carboxyl group through the phosphate of thephospholipid;

phthalidyl (shown in FIG. 1 of Clayton et al., Antimicrob. Agents Chemo.(1974) 5(6):670-671;

cyclic carbonates such as (5-R_(d)-2-oxo-1,3-dioxolen-4-yl)methyl esters(Sakamoto et al., Chem. Pharm. Bull. (1984) 32(6)2241-2248) where R_(d)is R₁, R₄ or aryl; and

The hydroxyl groups of the compounds of this invention optionally aresubstituted with one of groups III, IV or V disclosed in WO 94/21604, orwith isopropyl.

Table A lists examples of protecting group ester moieties that forexample can be bonded via oxygen to —C(O)O— and —P(O)(O—)₂ groups.Several amidates also are shown, which are bound directly to —C(O)— or—P(O)₂. Esters of structures 1-5, 8-10 and 16, 17, 19-22 are synthesizedby reacting the compound herein having a free hydroxyl with thecorresponding halide (chloride or acyl chloride and the like) andN,N-dicyclohexyl-N-morpholine carboxamidine (or another base such asDBU, triethylamine, CsCO₃, N,N-dimethylaniline and the like) in DMF (orother solvent such as acetonitrile or N-methylpyrrolidone). When thecompound to be protected is a phosphonate, the esters of structures 5-7,11, 12, 21, and 23-26 are synthesized by reaction of the alcohol oralkoxide salt (or the corresponding amines in the case of compounds suchas 13, 14 and 15) with the monochlorophosphonate or dichlorophosphonate(or another activated phosphonate).

TABLE A 1. —CH₂—C(O)—N(R₁)₂* 2. —CH₂—S(O)(R₁) 3. —CH₂—S(O)₂(R₁) 4.—CH₂—O—C(O)—CH₂—C₆H₅ 5. 3-cholesteryl 6. 3-pyridyl 7. N-ethylmorpholino8. —CH₂—O—C(O)—C₆H₅ 9. —CH₂—O—C(O)—CH₂CH₃ 10. —CH₂—O—C(O)—C(CH₃)₃ 11.—CH₂—CCl₃ 12. —C₆H₅ 13. —NH—CH₂—C(O)O—CH₂CH₃ 14.—N(CH₃)—CH₂—C(O)O—CH₂CH₃ 15. —NHR₁ 16. —CH₂—O—C(O)—C₁₀H₁₅ 17.—CH₂—O—C(O)—CH(CH₃)₂ 18. —CH₂—C#H(OC(O)CH₂R₁)—CH₂—(OC(O)CH₂R₁)* 19.

20.

21.

22.

23.

24.

25.

26.

#-chiral center is (R), (S) or racemate.

Other esters that are suitable for use herein are described in EP632048.

Protecting groups also includes “double ester” formingprofunctionalities such as —CH₂OC(O)OCH₃,

—CH₂SCOCH₃, —CH₂OCON(CH₃)₂, or alkyl- or aryl-acyloxyalkyl groups of thestructure —CH(R¹ or W⁵)O((CO)R³⁷) or —CH(R¹ or W⁵)((CO)OR³⁸) (linked tooxygen of the acidic group) wherein R³⁷ and R³⁸ are alkyl, aryl, oralkylaryl groups (see U.S. Pat. No. 4,968,788). Frequently R³⁷ and R³⁸are bulky groups such as branched alkyl, ortho-substituted aryl,meta-substituted aryl, or combinations thereof, including normal,secondary, iso- and tertiary alkyls of 1-6 carbon atoms. An example isthe pivaloyloxymethyl group. These are of particular use with prodrugsfor oral administration. Examples of such useful protecting groups arealkylacyloxymethyl esters and their derivatives, including—CH(CH₂CH₂OCH₃)OC(O)C(CH₃)₃,

—CH₂OC(O)C₁₀H₁₅, —CH₂OC(O)C(CH₃)₃, —CH(CH₂OCH₃)OC(O)C(CH₃)₃,—CH(CH(CH₃)₂)OC(O)C(CH₃)₃, —CH₂OC(O)CH₂CH(CH₃)₂, —CH₂OC(O)C₆H₁₁,—CH₂OC(O)C₆H₅, —CH₂OC(O)C₁₀H₁₅, —CH₂OC(O)CH₂CH₃, —CH₂OC(O)CH(CH₃)₂,—CH₂OC(O)C(CH₃)₃ and —CH₂OC(O)CH₂C₆H₅.

In some claims the protected acidic group is an ester of the acidicgroup and is the residue of a hydroxyl-containing functionality. Inother claims, an amino compound is used to protect the acidfunctionality. The residues of suitable hydroxyl or amino-containingfunctionalities are set forth above or are found in WO 95/07920. Ofparticular interest are the residues of amino acids, amino acid esters,polypeptides, or aryl alcohols. Typical amino acid, polypeptide andcarboxyl-esterified amino acid residues are described on pages 11-18 andrelated text of WO 95/07920 as groups L1 or L2. WO 95/07920 expresslyteaches the amidates of phosphonic acids, but it will be understood thatsuch amidates are formed with any of the acid groups set forth hereinand the amino acid residues set forth in WO 95/07920.

Typical esters for protecting acidic functionalities are also describedin WO 95/07920, again understanding that the same esters can be formedwith the acidic groups herein as with the phosphonate of the '920publication. Typical ester groups are defined at least on WO 95/07920pages 89-93 (under R³¹ or R³⁵), the table on page 105, and pages 21-23(as R). Of particular interest are esters of unsubstituted aryl such asphenyl or arylalkyl such benzyl, or hydroxy-, halo-, alkoxy-, carboxy-and/or alkylestercarboxy-substituted aryl or alkylaryl, especiallyphenyl, ortho-ethoxyphenyl, or C₁-C₄ alkylestercarboxyphenyl (salicylateC₁-C₁₂ alkylesters).

The protected acidic groups, particularly when using the esters oramides of WO 95/07920, are useful as prodrugs for oral administration.However, it is not essential that the acidic group be protected in orderfor the compounds of this invention to be effectively administered bythe oral route. When the compounds of the invention having protectedgroups, in particular amino acid amidates or substituted andunsubstituted aryl esters are administered systemically or orally theyare capable of hydrolytic cleavage in vivo to yield the free acid.

One or more of the acidic hydroxyls are protected. If more than oneacidic hydroxyl is protected then the same or a different protectinggroup is employed, e.g., the esters may be different or the same, or amixed amidate and ester may be used.

Typical hydroxy protecting groups described in Greene (pages 14-118)include substituted methyl and alkyl ethers, substituted benzyl ethers,silyl ethers, esters including sulfonic acid esters, and carbonates. Forexample:

-   -   Ethers (methyl, t-butyl, allyl);    -   Substituted Methyl Ethers (Methoxymethyl, Methylthiomethyl,        t-Butylthiomethyl, (Phenyldimethylsilyl)methoxymethyl,        Benzyloxymethyl, p-Methoxybenzyloxymethyl,        (4-Methoxyphenoxy)methyl, Guaiacolmethyl, t-Butoxymethyl,        4-Pentenyloxymethyl, Siloxymethyl, 2-Methoxyethoxymethyl,        2,2,2-Trichloroethoxymethyl, Bis(2-chloroethoxy)methyl,        2-(Trimethylsilyl)ethoxymethyl, Tetrahydropyranyl,        3-Bromotetrahydropyranyl, Tetrahydropthiopyranyl,        1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl,        4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahydropthiopyranyl        S,S-Dioxido,        1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl,        1,4-Dioxan-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl,        2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl));    -   Substituted Ethyl Ethers (1-Ethoxyethyl,        1-(2-Chloroethoxy)ethyl, 1-Methyl-1-methoxyethyl,        1-Methyl-1-benzyloxyethyl, 1-Methyl-1-benzyloxy-2-fluoroethyl,        2,2,2-Trichloroethyl, 2-Trimethylsilylethyl,        2-(Phenylselenyl)ethyl,    -   p-Chlorophenyl, p-Methoxyphenyl, 2,4-Dinitrophenyl, Benzyl);    -   Substituted Benzyl Ethers (p-Methoxybenzyl, 3,4-Dimethoxybenzyl,        o-Nitrobenzyl, p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl,        p-Cyanobenzyl, p-Phenylbenzyl, 2- and 4-Picolyl,        3-Methyl-2-picolyl N-Oxido, Diphenylmethyl,        p,p′-Dinitrobenzhydryl, 5-Dibenzosuberyl, Triphenylmethyl,        α-Naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,        Di(p-methoxyphenyl)phenylmethyl, Tri(p-methoxyphenyl)methyl,        4-(4′-Bromophenacyloxy)phenyldiphenylmethyl,        4,4′,4″-Tris(4,5-dichlorophthalimidophenyl)methyl,        4,4′,4″-Tris(levulinoyloxyphenyl)methyl,        4,4′,4″-Tris(benzoyloxyphenyl)methyl,        3-(Imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl,        1,1-Bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-Anthryl,        9-(9-Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl,        1,3-Benzodithiolan-2-yl, Benzisothiazolyl S,S-Dioxido);    -   Silyl Ethers (Trimethylsilyl, Triethylsilyl, Triisopropylsilyl,        Dimethylisopropylsilyl, Diethylisopropylsilyl,        Dimethylthexylsilyl, t-Butyldimethylsilyl, t-Butyldiphenylsilyl,        Tribenzylsilyl, Tri-p-xylylsilyl, Triphenylsilyl,        Diphenylmethylsilyl, t-Butylmethoxyphenylsilyl);    -   Esters (Formate, Benzoylformate, Acetate, Choroacetate,        Dichloroacetate, Trichloroacetate, Trifluoroacetate,        Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate,        p-Chlorophenoxyacetate, p-poly-Phenylacetate,        3-Phenylpropionate, 4-Oxopentanoate (Levulinate),        4,4-(Ethylenedithio)pentanoate, Pivaloate, Adamantoate,        Crotonate, 4-Methoxycrotonate, Benzoate, p-Phenylbenzoate,        2,4,6-Trimethylbenzoate (Mesitoate));    -   Carbonates (Methyl, 9-Fluorenylmethyl, Ethyl,        2,2,2-Trichloroethyl, 2-(Trimethylsilyl)ethyl,        2-(Phenylsulfonyl)ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl,        Vinyl, Allyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl,        3,4-Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl        Thiocarbonate, 4-Ethoxy-1-naphthyl, Methyl Dithiocarbonate);    -   Groups With Assisted Cleavage (2-Iodobenzoate, 4-Azidobutyrate,        4-Nitro-4-methylpentanoate, o-(Dibromomethyl)benzoate,        2-Fommylbenzenesulfonate, 2-(Methylthiomethoxy)ethyl Carbonate,        4-(Methylthiomethoxy)butyrate,        2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters        (2,6-Dichloro-4-methylphenoxyacetate, 2,6-Dichloro-4-(1,1,3,3        tetramethylbutyl)phenoxyacetate,        2,4-Bis(1,1-dimethylpropyl)phenoxyacetate,        Chlorodiphenylacetate, Isobutyrate, Monosuccinate,        (E)-2-Methyl-2-butenoate (Tigloate),        o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, α-Naphthoate,        Nitrate, Alkyl N,N,N′,N′-Tetramethylphosphorodiamidate,        N-Phenylcarbamate, Borate, Dimethylphosphinothioyl,        2,4-Dinitrophenylsulfenate); and    -   Sulfonates (Sulfate, Methanesulfonate (Mesylate),        Benzylsulfonate, Tosylate).

Typical 1,2-diol protecting groups (thus, generally where two OH groupsare taken together with the protecting functionality) are described inGreene at pages 118-142 and include Cyclic Acetals and Ketals(Methylene, Ethylidene, 1-t-Butylethylidene, 1-Phenylethylidene,(4-Methoxyphenyl)ethylidene, 2,2,2-Trichloroethylidene, Acetonide(Isopropylidene), Cyclopentylidene, Cyclohexylidene, Cycloheptylidene,Benzylidene, p-Methoxybenzylidene, 2,4-Dimethoxybenzylidene,3,4-Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho Esters(Methoxymethylene, Ethoxymethylene, Dimethoxymethylene,1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene,α-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene Derivative,α-(N,N-Dimethylamino)benzylidene Derivative, 2-Oxacyclopentylidene);Silyl Derivatives (Di-t-butylsilylene Group,1,3-(1,1,3,3-Tetraisopropyldisiloxanylidene), andTetra-t-butoxydisiloxane-1,3-diylidene), Cyclic Carbonates, CyclicBoronates, Ethyl Boronate and Phenyl Boronate.

More typically, 1,2-diol protecting groups include those shown in TableB, still more typically, epoxides, acetonides, cyclic ketals and arylacetals.

TABLE B

wherein R⁹ is C₁—C₆ alkyl.

Amino Protecting Groups

Another set of protecting groups include any of the typical aminoprotecting groups described by Greene at pages 315-385. They include:

-   -   Carbamates: (methyl and ethyl, 9-fluorenylmethyl,        9(2-sulfo)fluorenylmethyl, 9-(2,7-dibromo)fluorenylmethyl,        2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl,        4-methoxyphenacyl);    -   Substituted Ethyl: (2,2,2-trichoroethyl, 2-trimethylsilylethyl,        2-phenylethyl, 1-(1-adamantyl)-1-methylethyl,        1,1-dimethyl-2-haloethyl, 1,1-dimethyl-2,2-dibromoethyl,        1,1-dimethyl-2,2,2-trichloroethyl,        1-methyl-1-(4-biphenylyl)ethyl,        1-(3,5-di-t-butylphenyl)-1-methylethyl, 2-(2′- and        4′-pyridyl)ethyl, 2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl,        1-adamantyl, vinyl, allyl, 1-isopropylallyl, cinnamyl,        4-nitrocinnamyl, 8-quinolyl, N-hydroxypiperidinyl, alkyldithio,        benzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl,        p-chlorobenzyl, 2,4-dichlorobenzyl, 4-methylsulfinylbenzyl,        9-anthrylmethyl, diphenylmethyl);    -   Groups With Assisted Cleavage: (2-methylthioethyl,        2-methylsulfonylethyl, 2-(p-toluenesulfonyl)ethyl,        [2-(1,3-dithianyl)]methyl, 4-methylthiophenyl,        2,4-dimethylthiophenyl, 2-phosphonioethyl,        2-triphenylphosphonioisopropyl, 1,1-dimethyl-2-cyanoethyl,        m-choro-p-acyloxybenzyl, p-(dihydroxyboryl)benzyl,        5-benzisoxazolylmethyl, 2-(trifluoromethyl)-6-chromonylmethyl);    -   Groups Capable of Photolytic Cleavage: (m-nitrophenyl,        3,5-dimethoxybenzyl, o-nitrobenzyl, 3,4-dimethoxy-6-nitrobenzyl,        phenyl(o-nitrophenyl)methyl); Urea-Type Derivatives        (phenothiazinyl-(10)-carbonyl,        N′-p-toluenesulfonylaminocarbonyl, N′-phenylaminothiocarbonyl);    -   Miscellaneous Carbamates: (t-amyl, S-benzyl thiocarbamate,        p-cyanobenzyl, cyclobutyl, cyclohexyl, cyclopentyl,        cyclopropylmethyl, p-decyloxybenzyl, diisopropylmethyl,        2,2-dimethoxycarbonylvinyl, o-(N,N-dimethylcarboxamido)benzyl,        1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl,        1,1-dimethylpropynyl, di(2-pyridyl)methyl, 2-furanylmethyl,        2-Iodoethyl, Isobornyl, Isobutyl, Isonicotinyl,        p-(p′-Methoxyphenylazo)benzyl, 1-methylcyclobutyl,        1-methylcyclohexyl, 1-methyl-1-cyclopropylmethyl,        1-methyl-1-(3,5-dimethoxyphenyl)ethyl,        1-methyl-1-(p-phenylazophenyl)ethyl, 1-methyl-1-phenylethyl,        1-methyl-1-(4-pyridyl)ethyl, phenyl, p-(phenylazo)benzyl,        2,4,6-tri-t-butylphenyl, 4-(trimethylammonium)benzyl,        2,4,6-trimethylbenzyl);    -   Amides: (N-formyl, N-acetyl, N-choroacetyl, N-trichoroacetyl,        N-trifluoroacetyl, N-phenylacetyl, N-3-phenylpropionyl,        N-picolinoyl, N-3-pyridylcarboxamide, N-benzoylphenylalanyl,        N-benzoyl, N-p-phenylbenzoyl);    -   Amides With Assisted Cleavage: (N-o-nitrophenylacetyl,        N-o-nitrophenoxyacetyl, N-acetoacetyl,        (N′-dithiobenzyloxycarbonylamino)acetyl,        N-3-(p-hydroxyphenyl)propionyl, N-3-(o-nitrophenyl)propionyl,        N-2-methyl-2-(o-nitrophenoxy)propionyl,        N-2-methyl-2-(o-phenylazophenoxy)propionyl, N-4-chlorobutyryl,        N-3-methyl-3-nitrobutyryl, N-o-nitrocinnamoyl,        N-acetylmethionine, N-o-nitrobenzoyl,        N-o-(benzoyloxymethyl)benzoyl, 4,5-diphenyl-3-oxazolin-2-one);    -   Cyclic Imide Derivatives: (N-phthalimide, N-dithiasuccinoyl,        N-2,3-diphenylmaleoyl, N-2,5-dimethylpyrrolyl,        N-1,1,4,4-tetramethyldisilylazacyclopentane adduct,        5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one,        5-substituted 1,3-dibenzyl-1,3-5-triazacyclohexan-2-one,        1-substituted 3,5-dinitro-4-pyridonyl);    -   N-Alkyl and N-Aryl Amines: (N-methyl, N-allyl,        N-[2-(trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl,        N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl), Quaternary        Ammonium Salts, N-benzyl, N-di(4-methoxyphenyl)methyl,        N-5-dibenzosuberyl, N-triphenylmethyl,        N-(4-methoxyphenyl)diphenylmethyl, N-9-phenylfluorenyl,        N-2,7-dichloro-9-fluorenylmethylene, N-ferrocenylmethyl,        N-2-picolylamine N′-oxide);    -   Imine Derivatives: (N-1,1-dimethylthiomethylene, N-benzylidene,        N-p-methoxybenzylidene, N-diphenylmethylene,        N-[(2-pyridyl)mesityl]methylene,        N,(N′,N′-dimethylaminomethylene, N,N′-isopropylidene,        N-p-nitrobenzylidene, N-salicylidene, N-5-chlorosalicylidene,        N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene);    -   Enamine Derivatives: (N-(5,5-dimethyl-3-oxo-1-cyclohexenyl));    -   N-Metal Derivatives (N-borane derivatives, N-diphenylborinic        acid derivatives, N-[phenyl(pentacarbonylchromium- or        -tungsten)]carbenzyl, N-copper or N-zinc chelate);    -   N—N Derivatives: (N-nitro, N-nitroso, N-oxide);    -   N—P Derivatives: (N-diphenylphosphinyl,        N-dimethylthiophosphinyl, N-diphenylthiophosphinyl, N-dialkyl        phosphoryl, N-dibenzyl phosphoryl, N-diphenyl phosphoryl);    -   N-Si Derivatives, N—S Derivatives, and N-Sulfenyl Derivatives:        (N-benzenesulfenyl, N-o-nitrobenzenesulfenyl,        N-2,4-dinitrobenzenesulfenyl, N-pentachlorobenzenesulfenyl,        N-2-nitro-4-methoxybenzenesulfenyl, N-triphenylmethylsulfenyl,        N-3-nitropyridinesulfenyl); and N-sulfonyl Derivatives        (N-p-toluenesulfonyl, N-benzenesulfonyl,        N-2,3,6-trimethyl-4-methoxybenzenesulfonyl,        N-2,4,6-trimethoxybenzenesulfonyl,        N-2,6-dimethyl-4-methoxybenzenesulfonyl,        N-pentamethylbenzenesulfonyl,        N-2,3,5,6,-tetramethyl-4-methoxybenzenesulfonyl,        N-4-methoxybenzenesulfonyl, N-2,4,6-trimethylbenzenesulfonyl,        N-2,6-dimethoxy-4-methylbenzenesulfonyl,        N-2,2,5,7,8-pentamethylchroman-6-sulfonyl, N-methanesulfonyl,        N-β-trimethylsilyethanesulfonyl, N-9-anthracenesulfonyl,        N-4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonyl,        N-benzylsulfonyl, N-trifluoromethylsulfonyl,        N-phenacylsulfonyl).

More typically, protected amino groups include carbamates and amides,still more typically, —NHC(O)R¹ or —N═CR¹N(R¹)₂. Another protectinggroup, also useful as a prodrug for amino or —NH(R⁵), is:

See for example Alexander, J. et al. (1996) J. Med. Chem. 39:480-486.

Amino Acid and Polypeptide Protecting Group and Conjugates

An amino acid or polypeptide protecting group of a compound of theinvention has the structure R¹⁵NHCH(R¹⁶)C(O)—, where R¹⁵ is H, an aminoacid or polypeptide residue, or R⁵, and R¹⁶ is defined below.

R¹⁶ is lower alkyl or lower alkyl (C₁-C₆) substituted with amino,carboxyl, amide, carboxyl ester, hydroxyl, C₆-C₇ aryl, guanidinyl,imidazolyl, indolyl, sulfhydryl, sulfoxide, and/or alkylphosphate. R¹⁰also is taken together with the amino acid a N to form a proline residue(R¹⁰=—CH₂)₃—). However, R¹⁰ is generally the side group of anaturally-occurring amino acid such as H, —CH₃, —CH(CH₃)₂,—CH₂—CH(CH₃)₂, —CHCH₃—CH₂—CH₃, —CH₂—C₆H₅, —CH₂CH₂—S—CH₃, —CH₂OH,—CH(OH)—CH₃, —CH₂—SH, —CH₂—C₆H₄OH, —CH₂—CO—NH₂, —CH₂—CH₂—CO—NH₂,—CH₂—COOH, —CH₂—CH₂—COOH, —(CH₂)₄—NH₂ and —(CH₂)₃—NH—C(NH₂)—NH₂. R¹⁰also includes 1-guanidinoprop-3-yl, benzyl, 4-hydroxybenzyl,imidazol-4-yl, indol-3-yl, methoxyphenyl and ethoxyphenyl.

Another set of protecting groups include the residue of anamino-containing compound, in particular an amino acid, a polypeptide, aprotecting group, —NHSO₂R, NHC(O)R, —N(R)₂, NH₂ or —NH(R)(H), wherebyfor example a carboxylic acid is reacted, i.e. coupled, with the amineto form an amide, as in C(O)NR₂. A phosphonic acid may be reacted withthe amine to form a phosphonamidate, as in —P(O)(OR)(NR₂).

In general, amino acids have the structure R¹⁷C(O)CH(R¹⁶)NH—, where R¹⁷is —OH, —OR, an amino acid or a polypeptide residue. Amino acids are lowmolecular weight compounds, on the order of less than about 1000 MW andwhich contain at least one amino or imino group and at least onecarboxyl group. Generally the amino acids will be found in nature, i.e.,can be detected in biological material such as bacteria or othermicrobes, plants, animals or man. Suitable amino acids typically arealpha amino acids, i.e. compounds characterized by one amino or iminonitrogen atom separated from the carbon atom of one carboxyl group by asingle substituted or unsubstituted alpha carbon atom. Of particularinterest are hydrophobic residues such as mono- or di-alkyl or arylamino acids, cycloalkylamino acids and the like. These residuescontribute to cell permeability by increasing the partition coefficientof the parental drug. Typically, the residue does not contain asulfhydryl or guanidino substituent.

Naturally-occurring amino acid residues are those residues foundnaturally in plants, animals or microbes, especially proteins thereof.Polypeptides most typically will be substantially composed of suchnaturally-occurring amino acid residues. These amino acids are glycine,alanine, valine, leucine, isoleucine, serine, threonine, cysteine,methionine, glutamic acid, aspartic acid, lysine, hydroxylysine,arginine, histidine, phenylalanine, tyrosine, tryptophan, proline,asparagine, glutamine and hydroxyproline. Additionally, unnatural aminoacids, for example, valanine, phenylglycine and homoarginine are alsoincluded. Commonly encountered amino acids that are not gene-encoded mayalso be used in the present invention. All of the amino acids used inthe present invention may be either the D- or L-optical isomer. Inaddition, other peptidomimetics are also useful in the presentinvention. For a general review, see Spatola, A. F., in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983).

When protecting groups are single amino acid residues or polypeptidesthey optionally are substituted at R³ of substituents A¹, A² or A³ in acompound of the invention. These conjugates are produced by forming anamide bond between a carboxyl group of the amino acid (or C-terminalamino acid of a polypeptide for example). Similarly, conjugates areformed between R and an amino group of an amino acid or polypeptide.Generally, only one of any site in the parental molecule is amidatedwith an amino acid as described herein, although it is within the scopeof this invention to introduce amino acids at more than one permittedsite. Usually, a carboxyl group of R³ is amidated with an amino acid. Ingeneral, the α-amino or α-carboxyl group of the amino acid or theterminal amino or carboxyl group of a polypeptide are bonded to theparental functionalities, i.e., carboxyl or amino groups in the aminoacid side chains generally are not used to form the amide bonds with theparental compound (although these groups may need to be protected duringsynthesis of the conjugates as described further below).

With respect to the carboxyl-containing side chains of amino acids orpolypeptides it will be understood that the carboxyl group optionallywill be blocked, e.g., by R¹, esterified with R⁵ or amidated. Similarly,the amino side chains R¹⁶ optionally will be blocked with R¹ orsubstituted with R⁵.

Such ester or amide bonds with side chain amino or carboxyl groups, likethe esters or amides with the parental molecule, optionally arehydrolyzable in vivo or in vitro under acidic (pH<3) or basic (pH>10)conditions. Alternatively, they are substantially stable in thegastrointestinal tract of humans but are hydrolyzed enzymatically inblood or in intracellular environments. The esters or amino acid orpolypeptide amidates also are useful as intermediates for thepreparation of the parental molecule containing free amino or carboxylgroups. The free acid or base of the parental compound, for example, isreadily formed from the esters or amino acid or polypeptide conjugatesof this invention by conventional hydrolysis procedures.

When an amino acid residue contains one or more chiral centers, any ofthe D, L, meso, threo or erythro (as appropriate) racemates, scalematesor mixtures thereof may be used. In general, if the intermediates are tobe hydrolyzed non-enzymatically (as would be the case where the amidesare used as chemical intermediates for the free acids or free amines), Disomers are useful. On the other hand, L isomers are more versatilesince they can be susceptible to both non-enzymatic and enzymatichydrolysis, and are more efficiently transported by amino acid ordipeptidyl transport systems in the gastrointestinal tract.

Examples of suitable amino acids whose residues are represented by R^(x)or R^(y) include the following:

Glycine;

Aminopolycarboxylic acids, e.g., aspartic acid, β-hydroxyaspartic acid,glutamic acid, β-hydroxyglutamic acid, β-methylaspartic acid,β-methylglutamic acid, β,β-dimethylaspartic acid, γ-hydroxyglutamicacid, β,γ-dihydroxyglutamic acid, β-phenylglutamic acid,γ-methyleneglutamic acid, 3-aminoadipic acid, 2-aminopimelic acid,2-aminosuberic acid and 2-aminosebacic acid;

Amino acid amides such as glutamine and asparagine;

Polyamino- or polybasic-monocarboxylic acids such as arginine, lysine,β-aminoalanine, γ-aminobutyrine, ornithine, citruline, homoarginine,homocitrulline, hydroxylysine, allohydroxylsine and diaminobutyric acid;

Other basic amino acid residues such as histidine;

Diaminodicarboxylic acids such as α,α′-diaminosuccinic acid,α,α′-diaminoglutaric acid, α,α′-diaminoadipic acid, α,α′-diaminopimelicacid, α,α′-diamino-β-hydroxypimelic acid, α,α′-diaminosuberic acid,α,α′-diaminoazelaic acid, and α,α′-diaminosebacic acid;

Imino acids such as proline, hydroxyproline, allohydroxyproline,γ-methylproline, pipecolic acid, 5-hydroxypipecolic acid, andazetidine-2-carboxylic acid;

A mono- or di-alkyl (typically C₁-C₈ branched or normal) amino acid suchas alanine, valine, leucine, allylglycine, butyrine, norvaline,norleucine, heptyline, α-methylserine, α-amino-α-methyl-γ-hydroxyvalericacid, α-amino-α-methyl-δ-hydroxyvaleric acid,α-amino-α-methyl-ε-hydroxycaproic acid, isovaline, α-methylglutamicacid, α-aminoisobutyric acid, α-aminodiethylacetic acid,α-aminodiisopropylacetic acid, α-aminodi-n-propylacetic acid,α-aminodiisobutylacetic acid, α-aminodi-n-butylacetic acid,α-aminoethylisopropylacetic acid, α-amino-n-propylacetic acid,α-aminodiisoamyacetic acid, α-methylaspartic acid, α-methylglutamicacid, 1-aminocyclopropane-1-carboxylic acid, isoleucine, alloisoleucine,tert-leucine, β-methyltryptophan and α-amino-β-ethyl-α-phenylpropionicacid;

β-phenylserinyl;

Aliphatic α-amino-β-hydroxy acids such as serine, β-hydroxyleucine,β-hydroxynorleucine, β-hydroxynorvaline, and α-amino-β-hydroxystearicacid;

α-Amino, α-, γ-, δ- or ε-hydroxy acids such as homoserine,δ-hydroxynorvaline, γ-hydroxynorvaline and ε-hydroxynorleucine residues;canavine and canaline; γ-hydroxyomithine;

-   2-hexosaminic acids such as D-glucosaminic acid or D-galactosaminic    acid;-   α-Amino-β-thiols such as penicillamine, β-thiolnorvaline or    β-thiolbutyrine;

Other sulfur containing amino acid residues including cysteine;homocystine, β-phenylmethionine, methionine, S-allyl-L-cysteinesulfoxide, 2-thiolhistidine, cystathionine, and thiol ethers of cysteineor homocysteine;

Phenylalanine, tryptophan and ring-substituted α-amino acids such as thephenyl- or cyclohexylamino acids α-aminophenylacetic acid,α-aminocyclohexylacetic acid and α-amino-β-cyclohexylpropionic acid;phenylalanine analogues and derivatives comprising aryl, lower alkyl,hydroxy, guanidino, oxyalkylether, nitro, sulfur or halo-substitutedphenyl (e.g., tyrosine, methyltyrosine and o-chloro-, p-chloro-,3,4-dichloro, o-, m- or p-methyl-, 2,4,6-trimethyl-, 2-ethoxy-5-nitro-,2-hydroxy-5-nitro- and p-nitro-phenylalanine); furyl-, thienyl-,pyridyl-, pyrimidinyl-, purinyl- or naphthyl-alanines; and tryptophananalogues and derivatives including kynurenine, 3-hydroxykynurenine,2-hydroxytryptophan and 4-carboxytryptophan;

α-Amino substituted amino acids including sarcosine (N-methylglycine),N-benzylglycine, N-methylalanine, N-benzylalanine,N-methylphenylalanine, N-benzylphenylalanine, N-methylvaline andN-benzylvaline; and

α-Hydroxy and substituted α-hydroxy amino acids including serine,threonine, allothreonine, phosphoserine and phosphothreonine.

Polypeptides are polymers of amino acids in which a carboxyl group ofone amino acid monomer is bonded to an amino or imino group of the nextamino acid monomer by an amide bond. Polypeptides include dipeptides,low molecular weight polypeptides (about 1500-5000 MW) and proteins.Proteins optionally contain 3, 5, 10, 50, 75, 100 or more residues, andsuitably are substantially sequence-homologous with human, animal, plantor microbial proteins. They include enzymes (e.g., hydrogen peroxidase)as well as immunogens such as KLH, or antibodies or proteins of any typeagainst which one wishes to raise an immune response. The nature andidentity of the polypeptide may vary widely.

The polypeptide amidates are useful as immunogens in raising antibodiesagainst either the polypeptide (if it is not immunogenic in the animalto which it is administered) or against the epitopes on the remainder ofthe compound of this invention.

Antibodies capable of binding to the parental non-peptidyl compound areused to separate the parental compound from mixtures, for example indiagnosis or manufacturing of the parental compound. The conjugates ofparental compound and polypeptide generally are more immunogenic thanthe polypeptides in closely homologous animals, and therefore make thepolypeptide more immunogenic for facilitating raising antibodies againstit. Accordingly, the polypeptide or protein may not need to beimmunogenic in an animal typically used to raise antibodies, e.g.,rabbit, mouse, horse, or rat, but the final product conjugate should beimmunogenic in at least one of such animals. The polypeptide optionallycontains a peptidolytic enzyme cleavage site at the peptide bond betweenthe first and second residues adjacent to the acidic heteroatom. Suchcleavage sites are flanked by enzymatic recognition structures, e.g., aparticular sequence of residues recognized by a peptidolytic enzyme.

Peptidolytic enzymes for cleaving the polypeptide conjugates of thisinvention are well known, and in particular include carboxypeptidases.Carboxypeptidases digest polypeptides by removing C-terminal residues,and are specific in many instances for particular C-terminal sequences.Such enzymes and their substrate requirements in general are well known.For example, a dipeptide (having a given pair of residues and a freecarboxyl terminus) is covalently bonded through its α-amino group to thephosphorus or carbon atoms of the compounds herein. In claims where W₁is phosphonate it is expected that this peptide will be cleaved by theappropriate peptidolytic enzyme, leaving the carboxyl of the proximalamino acid residue to autocatalytically cleave the phosphonoamidatebond.

Suitable dipeptidyl groups (designated by their single letter code) areAA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL, AK, AM, AF, AP, AS, AT, AW,AY, AV, RA, RR, RN, RD, RC, RE, RQ, RG, RH, RI, RL, RK, RM, RF, RP, RS,RT, RW, RY, RV, NA, NR, NN, ND, NC, NE, NQ, NG, NH, NI, NL, NK, NM, NF,NP, NS, NT, NR, NY, NV, DA, DR, DN, DD, DC, DE, DQ, DG, DH, DI, DL, DK,DM, DF, DP, DS, DT, DW, DY, DV, CA, CR, CN, CD, CC, CE, CQ, CG, CH, CI,CL, CK, CM, CF, CP, CS, CT, CW, CY, CV, EA, ER, EN, ED, EC, EE, EQ, EG,EH, EI, EL, EK, EM, EF, EP, ES, ET, EW, EY, EV, QA, QR, QN, QD, QC, QE,QQ, QG, QH, QI, QL, QK, QM, QF, QP, QS, QT, QW, QY, QV, GA, GR, GN, GD,GC, GE, GQ, GG, GH, GI, GL, GK, GM, GF, GP, GS, GT, GW, GY, GV, HA, HR,HN, HD, HC, HE, HQ, HG, HH, HI, HL, HK, HM, HF, HP, HS, HT, HW, HY, HV,IA, IR, IN, ID, IC, IE, IQ, IG, IH, II, IL, IK, IM, IF, IP, IS, IT, IW,IY, IV, LA, LR, LN, LD, LC, LE, LQ, LG, LH, LI, LL, LK, LM, LF, LP, LS,LT, LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, KL, KK, KM, KF,KP, KS, KT, KW, KY, KV, MA, MR, MN, MD, MC, ME, MQ, MG, MH, MI, ML, MK,MM, MF, MP, MS, MT, MW, MY, MV, FA, FR, FN, FD, FC, FE, FQ, FG, FH, FI,FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN, PD, PC, PE, PQ, PG,PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, PY, PV, SA, SR, SN, SD, SC, SE,SQ, SG, SH, SI, SL, SK, SM, SF, SP, SS, ST, SW, SY, SV, TA, TR, TN, TD,TC, TE, TQ, TG, TH, TI, TL, TK, TM, TF, TP, TS, TT, TW, TY, TV, WA, WR,WN, WD, WC, WE, WQ, WG, WH, WI, WL, WK, WM, WF, WP, WS, WT, WW, WY, WV,YA, YR, YN, YD, YC, YE, YQ, YG, YH, YI, YL, YK, YM, YF, YP, YS, YT, YW,YY, YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL, VK, VM, VF, VP, VS,VT, VW, VY and VV.

Tripeptide residues are also useful as protecting groups. When aphosphonate is to be protected, the sequence —X²⁰⁰-pro-X²⁰⁴- (where X²⁰⁰is any amino acid residue and X²⁰¹ is an amino acid residue, a carboxylester of proline, or hydrogen) will be cleaved by luminalcarboxypeptidase to yield X²⁰⁰ with a free carboxyl, which in turn isexpected to autocatalytically cleave the phosphonoamidate bond. Thecarboxy group of X²⁰¹ optionally is esterified with benzyl.

Dipeptide or tripeptide species can be selected on the basis of knowntransport properties and/or susceptibility to peptidases that can affecttransport to intestinal mucosal or other cell types. Dipeptides andtripeptides lacking an α-amino group are transport substrates for thepeptide transporter found in brush border membrane of intestinal mucosalcells (Bai, J. P. F., (1992) Pharm Res. 9:969-978). Transport competentpeptides can thus be used to enhance bioavailability of the amidatecompounds. Di- or tripeptides having one or more amino acids in the Dconfiguration are also compatible with peptide transport and can beutilized in the amidate compounds of this invention. Amino acids in theD configuration can be used to reduce the susceptibility of a di- ortripeptide to hydrolysis by proteases common to the brush border such asaminopeptidase N. In addition, di- or tripeptides alternatively areselected on the basis of their relative resistance to hydrolysis byproteases found in the lumen of the intestine. For example, tripeptidesor polypeptides lacking asp and/or glu are poor substrates foraminopeptidase A, di- or tripeptides lacking amino acid residues on theN-terminal side of hydrophobic amino acids (leu, tyr, phe, val, trp) arepoor substrates for endopeptidase, and peptides lacking a pro residue atthe penultimate position at a free carboxyl terminus are poor substratesfor carboxypeptidase P. Similar considerations can also be applied tothe selection of peptides that are either relatively resistant orrelatively susceptible to hydrolysis by cytosolic, renal, hepatic, serumor other peptidases. Such poorly cleaved polypeptide amidates areimmunogens or are useful for bonding to proteins in order to prepareimmunogens.

Specific Embodiments of the Invention

Specific values described for radicals, substituents, and ranges, aswell as specific embodiments of the invention described herein, are forillustration only; they do not exclude other defined values or othervalues within defined ranges.

In one specific embodiment of the invention, the conjugate is a compoundthat is substituted with one or more phosphonate groups either directlyor indirectly through a linker; and that is optionally substituted withone or more groups A⁰; or a pharmaceutically acceptable salt thereof,wherein:

A⁰ is A¹, A² or W³;

A¹ is:

A² is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;

R^(x) is independently H, R¹, W³, a protecting group, or the formula:

wherein:

R^(y) is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R² is independently H, R¹, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is R^(x), —N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y)R^(x), N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

R^(5a) is independently alkylene of 1 to 18 carbon atoms, alkenylene of2 to 18 carbon atoms, or alkynylene of 2-18 carbon atoms any one ofwhich alkylene, alkenylene or alkynylene is substituted with 0-3 R³groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(Y¹)W⁵, —SO₂R⁵, or —SO₂W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A3 groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1; and

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In another specific embodiment of the invention A¹ is of the formula:

In another specific embodiment of the invention A¹ is of the formula:

In another specific embodiment of the invention A¹ is of the formula:

In another specific embodiment of the invention A¹ is of the formula:

In another specific embodiment of the invention A¹ is of the formula:

and W⁵a is a carbocycle or a heterocycle where W⁵a is independentlysubstituted with 0 or 1 R² groups. A specific value for M12a is 1.

In another specific embodiment of the invention A¹ is of the formula:

In another specific embodiment of the invention A¹ is of the formula:

In another specific embodiment of the invention A¹ is of the formula:

wherein W^(5a) is a carbocycle independently substituted with 0 or 1 R²groups.

In another specific embodiment of the invention A¹ is of the formula:

wherein Y^(2b) is O or N(R²); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A¹ is of the formula:

wherein W^(5a) is a carbocycle independently substituted with 0 or 1 R²groups.

In another specific embodiment of the invention A¹ is of the formula:

wherein W^(5a) is a carbocycle or heterocycle where W^(5a) isindependently substituted with 0 or 1 R² groups.

In another specific embodiment of the invention A¹ is of the formula:

wherein Y^(2b) is O or N(R²); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In a specific embodiment of the invention A² is of the formula:

In another specific embodiment of the invention A² is of the formula:

In another specific embodiment of the invention M12b is 1.

In another specific embodiment of the invention e M12b is 0, Y² is abond and W⁵ is a carbocycle or heterocycle where W⁵ is optionally andindependently substituted with 1, 2, or 3 R² groups.

In another specific embodiment of the invention A2 is of the formula:

wherein W^(5a) is a carbocycle or heterocycle where W^(5a) is optionallyand independently substituted with 1, 2, or 3 R² groups.

In another specific embodiment of the invention M12a is 1.

In another specific embodiment of the invention A² is selected fromphenyl, substituted phenyl, benzyl, substituted benzyl, pyridyl andsubstituted pyridyl.

In another specific embodiment of the invention A² is of the formula:

In another specific embodiment of the invention A² is of the formula:

In another specific embodiment of the invention M12b is 1.

In a specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; and Y²a is O, N(R^(x)) or S.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R^(x)).

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R^(x)); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is or N(R^(x)); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention M12d is 1.

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention W⁵ is a carbocycle.

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention W⁵ is phenyl.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; and Y^(2a) is O, N(R^(x)) or S.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R^(x)).

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R^(x)); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention R¹ is H.

In another specific embodiment of the invention A³ is of the formula:

wherein the phenyl carbocycle is substituted with 0, 1, 2, or 3 R²groups.

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; and Y²a is O, N(R²) or S.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; Y^(2b) is O or N(R²); and Y^(2c) is O, N(R¹)or S.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; Y^(2b) is O or N(R); Y is O or N(R^(y)); andM12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R²); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R²).

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; and Y^(2a) is O, N(R²) or S.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; Y^(2b) is O or N(R²); and Y^(2c) is O,N(R^(y)) or S.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(1a) is O or S; Y^(2b) is O or N(R²); Y^(2d) is O or N(R^(y));and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R²); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R²).

In another specific embodiment of the invention A³ is of the formula:

wherein Y^(2b) is O or N(R^(x)); and M12d is 1, 2, 3, 4, 5, 6, 7 or 8.

In another specific embodiment of the invention A³ is of the formula:

wherein the phenyl carbocycle is substituted with 0, 1, 2, or 3 R²groups.

In another specific embodiment of the invention A³ is of the formula:

wherein the phenyl carbocycle is substituted with 0, 1, 2, or 3 R²groups.

In another specific embodiment of the invention A³ is of the formula:

In a specific embodiment of the invention A⁰ is of the formula:

wherein each R is independently (C₁-C₆)alkyl.

In a specific embodiment of the invention R^(x) is independently H, R¹,W³, a protecting group, or the formula:

wherein:

R^(y) is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R² is independently H, R¹, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups.

In a specific embodiment of the invention R^(x) is of the formula:

wherein Y^(1a) is O or S; and Y^(2c) is O, N(R^(y)) or S.

In a specific embodiment of the invention R^(x) is of the formula:

wherein Y^(1a) is O or S; and Y^(2d) is O or N(R^(y)).

In a specific embodiment of the invention R^(x) is of the formula:

In a specific embodiment of the invention R^(y) is hydrogen or alkyl of1 to 10 carbons.

In a specific embodiment of the invention R^(x) is of the formula:

In a specific embodiment of the invention R^(x) is of the formula:

In a specific embodiment of the invention R^(x) is of the formula:

In a specific embodiment of the invention Y¹ is O or S.

In a specific embodiment of the invention Y² is O, N(R^(y)) or S.

In one specific embodiment of the invention R^(x) is a group of theformula:

wherein:

m1a, m1b, m1c, m1d and m1e are independently 0 or 1;

m12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

R^(y) is H, W³, R² or a protecting group;

provided that:

if m1a, m12c, and mid are 0, then m1b, m1c and m1c are 0;

if m1a and m12c are 0 and m1d is not 0, then m1b and m1c are 0;

if m1a and mid are 0 and m12c is not 0, then m1b and at least one of m1cand m1e are 0;

if m1a is 0 and m12c and m1d are not 0, then m1b is 0;

if m12c and m1d are 0 and m1a is not 0, then at least two of m1b, m1cand m1e are 0;

if m12c is 0 and m1a and mid are not 0, then at least one of m1b and m1care 0; and

if m1d is 0 and m1a and m12c are not 0, then at least one of m1c and m1eare 0.

In another specific embodiment, the invention provides a compound of theformula:

[DRUG]-(A⁰)_(nn)

or a pharmaceutically acceptable salt thereof wherein,

DRUG is a compound of any one of formulae 501-569

nn is 1, 2, or 3;

A⁰ is A¹, A² or W³ with the proviso that the compound includes at leastone A¹;

A¹ is:

A² is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;

R^(x) is independently H, R¹, W³, a protecting group, or the formula:

wherein:

R^(y) is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R² is independently H, R¹, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b)R^(3c) or R^(3d), provided that when R³ is bound toa heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y;

R^(3c) is —R^(x), —N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), —N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

R^(5a) is independently alkylene of 1 to 18 carbon atoms, alkenylene of2 to 18 carbon atoms, or alkynylene of 2-18 carbon atoms any one ofwhich alkylene, alkenylene or alkynylene is substituted, with 0-3 R³groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(Y¹)W⁵, —SO₂R⁵, or —SO₂W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A³ groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1; and

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In another specific embodiment the invention provides a compound of anyone of formulae 1-108 wherein:

A⁰ is A¹;

A¹ is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;

R^(x) is independently H, W³, a protecting group, or the formula:

R^(y) is independently H, W³, R² or a protecting group;

R¹ is independently H or alkyl of 1 to 18 carbon atoms;

R² is independently H, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups;

R^(3a) is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ isbound to a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is R^(x), N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), —N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R¹ groups;

R^(5a) is independently alkylene of 1 to 18 carbon atoms, alkenylene of2 to 18 carbon atoms, or alkynylene of 2-18 carbon atoms any one ofwhich alkylene, alkenylene or alkynylene is substituted with 0-3 R³groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(Y)W⁵, —SO₂R⁵, or —SO₂W;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A³ groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1; and

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In another specific embodiment, the invention provides a compound of theformula:

[DRUG]-[L-P(═Y¹)—Y²—R^(x)]_(nn)

or a pharmaceutically acceptable salt thereof wherein,

DRUG is a compound of any one of 501-569;

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;

R^(x) is independently H, W³, a protecting group, or the formula:

R^(y) is independently H, W³, R² or a protecting group;

R² is independently H, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is R^(x), —N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), —N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(Y¹)W⁵, —SO₂R⁵, or —SO₂W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

M2 is 1, 2, or 3;

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

nn is 1, 2, or 3; and

L is a linking group.

In another specific embodiment, the invention provides a compound ofwhich is a compound of the formula:

[DRUG]-(A⁰)_(nn)

or a pharmaceutically acceptable salt thereof wherein,

DRUG is a compound of any one of formulae 501-569;

nn is 1, 2, or 3;

A⁰ is A¹, A², or W³ with the proviso that the compound includes at leastone A¹;

A¹ is:

A² is:

A³ is:

Y¹ is independently O, S, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), or N(N(R^(x))(R^(x)));

Y² is independently a bond, O, N(R^(x)), N(O)(R^(x)), N(OR^(x)),N(O)(OR^(x)), N(N(R^(x))(R^(x))), —S(O)_(M2)—, or —S(O)_(M2)—S(O)_(M2)—;

R^(x) is independently H, W³, a protecting group, or the formula:

R^(y) is independently H, W³, R² or a protecting group;

R² is independently H, R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is F, Cl, Br, I, —CN, N₃ or —NO₂;

R^(3b) is Y¹;

R^(3c) is —R^(x), N(R^(x))(R^(x)), —SR^(x), —S(O)R^(x), —S(O)₂R^(x),—S(O)(OR^(x)), —S(O)₂(OR^(x)), —OC(Y¹)R^(x), —OC(Y¹)OR^(x),—OC(Y¹)(N(R^(x))(R^(x))), —SC(Y¹)R^(x), —SC(Y¹)OR^(x),—SC(Y¹)(N(R^(x))(R^(x))), —N(R^(x))C(Y¹)R^(x), —N(R^(x))C(Y¹)OR^(x), or—N(R^(x))C(Y¹)(N(R^(x))(R^(x)));

R^(3d) is —C(Y¹)R^(x), —C(Y¹)OR^(x) or —C(Y¹)(N(R^(x))(R^(x)));

R⁴ is an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms,or alkynyl of 2 to 18 carbon atoms;

R⁵ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

W³ is W⁴ or W⁵;

W⁴ is R⁵, —C(Y¹)R⁵, —C(yl)W⁵, —SO₂R⁵, or —SO₂W⁵;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups;

W⁶ is W³ independently substituted with 1, 2, or 3 A³ groups;

M2 is 0, 1 or 2;

M12a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M12b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

M1a, M1c, and M1d are independently 0 or 1; and

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In compounds of the invention W⁵ carbocycles and W⁵ heterocycles may beindependently substituted with 0 to 3 R² groups. W⁵ may be a saturated,unsaturated or aromatic ring comprising a mono- or bicyclic carbocycleor heterocycle. W⁵ may have 3 to 10 ring atoms, e.g., 3 to 7 ring atoms.The W⁵ rings are saturated when containing 3 ring atoms, saturated ormono-unsaturated when containing 4 ring atoms, saturated, or mono- ordi-unsaturated when containing 5 ring atoms, and saturated, mono- ordi-unsaturated, or aromatic when containing 6 ring atoms.

A W⁵ heterocycle may be a monocycle having 3 to 7 ring members (2 to 6carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or abicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S). W⁵ heterocyclic monocyclesmay have 3 to 6 ring atoms (2 to 5 carbon atoms and 1 to 2 heteroatomsselected from N, O, and S); or 5 or 6 ring atoms (3 to 5 carbon atomsand 1 to 2 heteroatoms selected from N and S). W⁵ heterocyclic bicycleshave 7 to 10 ring atoms (6 to 9 carbon atoms and 1 to 2 heteroatomsselected from N, O, and S) arranged as a bicyclo [4,5], [5,5, [5,6], or[6,6] system; or 9 to 10 ring atoms (8 to 9 carbon atoms and 1 to 2hetero atoms selected from N and S) arranged as a bicyclo [5,6] or [6,6]system. The W⁵ heterocycle may be bonded to Y² through a carbon,nitrogen, sulfur or other atom by a stable covalent bond.

W⁵ heterocycles include for example, pyridyl, dihydropyridyl isomers,piperidine, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl,imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, furanyl,thiofuranyl, thienyl, and pyrrolyl. W⁵ also includes, but is not limitedto, examples such as:

W⁵ carbocycles and heterocycles may be independently substituted with 0to 3 R² groups, as defined above. For example, substituted W⁵carbocycles include:

Examples of substituted phenyl carbocycles include:

Conjugates of Formula I

In one embodiment, the invention provides a conjugate of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

B is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrmidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O⁶-methylguanine, N⁶-methyladenine,O⁴-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole,substituted triazole, and pyrazolo[3,4-D]pyrimidine;

X is selected from O, C(R^(y))₂, C═C(R^(y))₂, NR and S;

Z¹ is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I;

Z² is selected from H, C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, and C₁-C₈ substitutedalkynyl,

Y¹ is independently O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;

Y² is independently a bond, O, CR₂, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR),N—NR₂, S, S—S, S(O), or S(O)₂;

M2 is 0, 1 or 2;

R^(y) is independently H, F, Cl, Br, I, OH, R, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₈ alkyl, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, alkylsulfone (—SO₂R), arylsulfone(—SO₂Ar), arylsulfoxide (—SOAr), arylthio (—SAr), sulfonamide (—SO₂NR₂),alkylsulfoxide (—SOR), ester (—C(═O)OR), amido (—C(═O)NR₂), 5-7 memberedring lactam, 5-7 membered ring lactone, nitrile (—CN), azido (—N₃),nitro (—NO₂), C₁-C₈ alkoxy (—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl,C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, polyethyleneoxy, aprotecting group (PG), or W³; or when taken together, R^(y) forms acarbocyclic ring of 3 to 7 carbon atoms;

R^(x) is independently R^(y), a protecting group, or the formula:

wherein:

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and

R is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₈substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, or a protecting group; and

W³ is W⁴ or W⁵, where W⁴ is R, —C(Y¹)R^(y), —C(Y¹)W⁵, —SO₂R^(y), or—SO₂W⁵; and W⁵ is a carbocycle or a heterocycle wherein W⁵ isindependently substituted with 0 to 3 R^(y) groups.

For a conjugate of Formula I, in one specific embodiment, C₁-C₈substituted alkyl, C₁-C₈ substituted alkenyl, C₁-C₈ substituted alkynyl,C₆-C₂₀ substituted aryl, and C₂-C₂₀ substituted heterocycle areindependently substituted with one or more substituents selected from F,Cl, Br, I, OH, —NH₂, —NH₃ ⁺, —NHR, —NR₂, —NR₃ ⁺, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, —SO₂R, —SO₂Ar, —SOAr, —SAr, —SO₂NR₂,—SOR, —CO₂R, —C(═O)NR₂, 5-7 membered ring lactam, 5-7 membered ringlactone, —CN, —N₃, —NO₂, C₁-C₈ alkoxy, C₁-C₈ trifluoroalkyl, C₁-C₈alkyl, C₃-C₁₂ carbocycle, C₆-C₂₀ aryl, C₂-C₂₀ heterocycle,polyethyleneoxy, phosphonate, phosphate, and a prodrug moiety.

For a conjugate of Formula I, in one specific embodiment, “protectinggroup” is selected from a carboxyl ester, a carboxamide, an aryl ether,an alkyl ether, a trialkylsilyl ether, a sulfonic acid ester, acarbonate, and a carbamate.

For a conjugate of Formula I, in one specific embodiment, W⁵ is selectedfrom the structures:

For a conjugate of Formula I, in one specific embodiment, X is O andR^(y) is H.

For a conjugate of Formula I, in one specific embodiment, X is C═CH₂ andR^(y) is H.

For a conjugate of Formula I, in one specific embodiment, Z¹ is OH.

For a conjugate of Formula I, in one specific embodiment, Z² is C₁-C₈alkyl or C₁-C₈ substituted alkyl.

For a conjugate of Formula I, in one specific embodiment, Z² is CH₃.

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

wherein, in a more specific embodiment, Z¹ is OH; Z² is C₁-C₈ alkyl orC₁-C₈ substituted alkyl; and Z² is CH₃.

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

wherein R² is H or C₁-C₈ alkyl.

In one specific embodiment, the conjugate of formula I has the followingformula:

In one specific embodiment, the conjugate of formula I has the followingformula:

wherein Y^(2c) is O, N(R^(y)) or S.

In one specific embodiment, the conjugate of formula I has the followingformula:

wherein, in a more specific embodiment, Y^(2c) is O; Y^(2c) is N(CH₃);and R^(y) is H or C₁-C₈ alkyl.

For a conjugate of Formula I, in one specific embodiment, thesubstituted triazole has the structure:

In one specific embodiment, the conjugate of Formula I is a conjugate ofthe following formula:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

B is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-D]pyrimidine;

Z¹ is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I;

Z² is selected from H, C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, and C₁-C₈ substitutedalkynyl,

R^(y) is independently H, F, Cl, Br, I, OH, R, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₈ alkyl, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, alkylsulfone (—SO₂R), arylsulfone(—SO₂Ar), arylsulfoxide (—SOAr), arylthio (—SAr), sulfonamide (—SO₂NR₂),alkylsulfoxide (—SOR), ester (—C(═O)OR), amido (—C(═O)NR₂), 5-7 memberedring lactam, 5-7 membered ring lactone, nitrile (—CN), azido (—N₃),nitro (—NO₂), C₁-C₈ alkoxy (—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl,C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, polyethyleneoxy, aprotecting group (PG), or W³; or when taken together, R^(y) forms acarbocyclic ring of 3 to 7 carbon atoms;

R is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₈substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, or a protecting group; and

W³ is W⁴ or W⁵, where W⁴ is R, —C(Y¹)R^(y), —C(Y¹)W⁵, —SO₂R^(y), or—SO₂W⁵; and W⁵ is a carbocycle or a heterocycle wherein W⁵ isindependently substituted with 0 to 3 R^(y) groups.

In one specific embodiment, the conjugate of Formula I has the followingformula:

wherein PG is a protecting group selected from an ether-forming group, athioether-forming group, an ester-forming group, a thioester-forminggroup, a silyl-ether forming group, an amide-forming group, anacetal-forming group, a ketal-forming group, a carbonate-forming group,a carbamate-forming group, a urea-forming group, an amino acidconjugate, and a polypeptide conjugate.

In one specific embodiment, the invention provides a conjugate ofFormula I having one of the following formulae:

or a pharmaceutically acceptable salt or solvate thereof; wherein B isadenine, guanine, cytosine, uracil, thymine, 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine,nebularine, nitropyrrole, nitroindole, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O⁶-methylguanine, N⁶-methyladenine,O⁴-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole,substituted triazole, or pyrazolo[3,4-D]pyrimidine. In an additionalembodiment, the compound is isolated and purified.

In one specific embodiment, the invention provides a conjugate ofFormula I having one of the following formulae:

or a pharmaceutically acceptable salt or solvate thereof; wherein B isadenine, guanine, cytosine, uracil, thymine, 7-deazaadenine,2,6-diaminopurine, 5-fluorocytosine, or c-propyl-2,6-diaminopurine. Inan additional embodiment, the compound is isolated and purified.

In one specific embodiment, the invention provides a conjugate ofFormula I having one of the following formulae:

or a pharmaceutically acceptable salt or solvate thereof. In anadditional embodiment, the compound is isolated and purified.

In one specific embodiment, the invention provides a conjugate ofFormula I having one of the following formulae:

or a pharmaceutically acceptable salt or solvate thereof; wherein B isadenine, guanine, cytosine, uracil, thymine, 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine,nebularine, nitropyrrole, nitroindole, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine,4-thiothymine, 4-thiouracil, O⁶-methylguanine, N⁶-methyladenine,O⁴-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole,substituted triazole, or pyrazolo[3,4-D]pyrimidine. In an additionalembodiment, the compound is isolated and purified.

In one specific embodiment, the invention provides a conjugate ofFormula I having one of the following formulae:

or a pharmaceutically acceptable salt or solvate thereof; wherein B isadenine, guanine, cytosine, uracil, thymine, 7-deazaadenine,2,6-diaminopurine, 5-fluorocytosine, or c-propyl-2,6-diaminopurine. Inan additional embodiment, the compound is isolated and purified.

In one specific embodiment, the invention provides a conjugate ofFormula I having one of the following formulae:

or a pharmaceutically acceptable salt or solvate thereof. In anadditional embodiment, the compound is isolated and purified.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of HCV infection inan infected animal comprising administering to said animal, apharmaceutical composition or formulation comprising an effective amountof a conjugate of formula I, or a pharmaceutically acceptable salt orsolvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of HCV infection inan infected animal comprising administering to said animal apharmaceutical composition or formulation comprising a conjugate offormula I, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of HCV infection inan infected animal comprising administering said animal with apharmaceutical combination composition or formulation comprising aneffective amount of a conjugate of formula I, or a pharmaceuticallyacceptable salt or solvate thereof, and a second compound havinganti-HCV properties.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of formula I,or a pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable excipient.

In one embodiment, the invention also provides a method of inhibiting aviral enzyme comprising the step of contacting a sample suspected ofcontaining viral infected cells or tissues with a conjugate of formulaI, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of a viral infectionin an animal which comprises administering to said animal a formulationcomprising a therapeutically effective amount of a conjugate of formulaI, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides the use of a conjugate offormula I, or a pharmaceutically acceptable salt or solvate thereof toprepare a medicament for treatment of HCV.

In one embodiment, the invention also provides a conjugate of formula I,or a pharmaceutically acceptable salt or solvate thereof, which iscapable of accumulating in human PBMC.

In one embodiment, the invention also provides a conjugate wherein thebioavailability of the conjugate or an intracellular metabolite of theconjugate in human PBMC is improved compared to the corresponding analoglacking the phosphonate group. For example, in one embodiment, thehalf-life is improved by at least about 50%; in another embodiment, thehalf-life is improved by at least about 100%; and in another embodiment,the half-life is improved by greater than 100%.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of formula I,or a pharmaceutically acceptable salt or solvate thereof, apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an AIDS treatment agent selected from an HIV inhibitor agent,an anti-infective agent, and an immunomodulator.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula I,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an HIV-protease inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula I,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of a reverse transcriptase inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula I,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of a non-nucleoside reverse transcriptase inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula I,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an HIV integrase inhibitor.

In one embodiment, the invention also provides a process for making apharmaceutical composition comprising combining a conjugate of formulaI, or a pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable excipient.

In one embodiment, the invention also provides a method of inhibitingRNA-dependent RNA polymerase comprising administering to a mammal inneed of such treatment, a therapeutically effective amount of aconjugate of Formula I, or a pharmaceutically acceptable salt or solvatethereof.

In one embodiment, the invention also provides a method of treating anHCV infection comprising administering to a mammal in need of suchtreatment a therapeutically effective amount of a conjugate of formulaI, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method of treating adisorder affecting white blood cells comprising: administering aconjugate of Formula I, or a pharmaceutically acceptable salt or solvatethereof to a patient in need of white-blood cell targeting.

In one embodiment, the invention also provides a method of manufacturingan HCV inhibitor conjugate having both selectivity for white blood cellsand a desired pharmaceutical activity, comprising: chemicallysynthesizing a conjugate of Formula I (as described herein), whereinsaid conjugate differs from a second structure of a compound known tohave said desired pharmaceutical activity by having at least onehydrogen atom of said second structure replaced by an organicsubstituent comprising a prodrug moiety or incipient prodrug moiety.

In one embodiment, the invention also provides a method of accumulatingan RNA-dependent RNA polymerase inhibitor compound inside a white bloodcell, comprising administering to a sample, a composition comprising aconjugate of formula I, or a pharmaceutically acceptable salt or solvatethereof. In one specific embodiment, said sample is a patient.

Conjugates of Formula II

In one embodiment, the invention provides a conjugate of Formula II:

or a pharmaceutically acceptable salt or solvate thereof; wherein:

B is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-D]pyrimidine;

X is selected from O, C(R^(y))₂, OC(R^(y))₂, NR and S;

Z¹ is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I;

Z² is selected from H, C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, and C₁-C₈ substitutedalkynyl,

Y¹ is independently O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;

Y² is independently a bond, O, CR₂, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR),N—NR₂, S, S—S, S(O), or S(O)₂;

M2 is 0, 1 or 2;

R^(y) is independently H, F, Cl, Br, I, OH, R, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₈ alkyl, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, alkylsulfone (—SO₂R), arylsulfone(—SO₂Ar), arylsulfoxide (—SOAr), arylthio (—SAr), sulfonamide (—SO₂NR₂),alkylsulfoxide (—SOR), ester (—C(═O)OR), amido (—C(═O)NR₂), 5-7 memberedring lactam, 5-7 membered ring lactone, nitrile (—CN), azido (—N₃),nitro (—NO₂), C₁-C₈ alkoxy (—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl,C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, polyethyleneoxy, aprotecting group, or W³; or when taken together, R^(y) forms acarbocyclic ring of 3 to 7 carbon atoms;

R^(x) is independently R^(y), a protecting group, or the formula:

wherein:

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and

R is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₉substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, or a protecting group; and

W³ is W⁴ or W⁵, where W⁴ is R, —C(Y¹)R^(y), —C(Y¹)W⁵, —SO₂R^(y), or—SO₂W⁵; and W⁵ is a carbocycle or a heterocycle wherein W⁵ isindependently substituted with 0 to 3 R^(y) groups.

For a conjugate of Formula II, in one specific embodiment, C₁-C₈substituted alkyl, C₁-C₈ substituted alkenyl, C₁-C₈ substituted alkynyl,C₆-C₂₀ substituted aryl, and C₂-C₂₀ substituted heterocycle areindependently substituted with one or more substituents selected from F,Cl, Br, I, OH, —NH₂, —NH₃ ⁺, —NHR, —NR₂, —NR₃ ⁺, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, —SO₂R, —SO₂Ar, —SOAr, —SAr, —SO₂NR₂,—SOR, —CO₂R, —C(═O)NR₂, 5-7 membered ring lactam, 5-7 membered ringlactone, —CN, —N₃, —NO₂, C₁-C₈ alkoxy, C₁-C₈ trifluoroalkyl, C₁-C₉alkyl, C₃-C₁₂ carbocycle, C₆-C₂₀ aryl, C₂-C₂₀ heterocycle,polyethyleneoxy, phosphonate, phosphate, and a prodrug moiety.

For a conjugate of Formula II, in one specific embodiment, “protectinggroup” is selected from a carboxyl ester, a carboxamide, an aryl ether,an alkyl ether, a trialkylsilyl ether, a sulfonic acid ester, acarbonate, and a carbamate.

For a conjugate of Formula II, in one specific embodiment, W⁵ isselected from the structures:

For a conjugate of Formula II, in one specific embodiment, X is O andR^(y) is H.

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein, in a more specific embodiment, Z¹ is OH; and Z² is CH₃.

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein, in a more specific embodiment, Z² is C₁-C₈ alkyl or C₁-C₉substituted alkyl.

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein R² is H or C₁-C₈ alkyl.

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein Y^(2c) is O, N(R^(y)) or S.

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein, in a more specific embodiment, Y^(2c) is O; Y^(2c) is N(CH₃).

In one specific embodiment, the substituted triazole has the structure:

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein:

B is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-D]pyrimidine;

X^(a) is selected from O, NR and S;

Z¹ is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F,Cl, Br, and I;

Z² is selected from H, C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, and C₁-C₈ substitutedalkynyl,

R^(y) is independently H, F, Cl, Br, I, OH, R, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₈ alkyl, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, alkylsulfone (—SO₂R), arylsulfone(—SO₂Ar), arylsulfoxide (—SOAr), arylthio (—SAr), sulfonamide (—SO₂NR₂),alkylsulfoxide (—SOR), ester (—C(═O)OR), amido (—C(═O)NR₂), 5-7 memberedring lactam, 5-7 membered ring lactone, nitrile (—CN), azido (—N₃),nitro (—NO₂), C₁-C₈ alkoxy (—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl,C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₉substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, polyethyleneoxy, aprotecting group, or W³; or when taken together, R^(y) forms acarbocyclic ring of 3 to 7 carbon atoms;

R is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₈substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, or a protecting group; and

W³ is W⁴ or W⁵, where W⁴ is R, —C(Y¹)R^(y), —C(Y¹)W⁵, —SO₂R^(y), or—SO₂W⁵; and W⁵ is a carbocycle or a heterocycle wherein W⁵ isindependently substituted with 0 to 3 R^(y) groups.

In one specific embodiment, the conjugate of Formula II has thefollowing formula:

wherein PG is a protecting group selected from an ether-forming group, athioether-forming group, an ester-forming group, a thioester-forminggroup, a silyl-ether forming group, an amide-forming group, anacetal-forming group, a ketal-forming group, a carbonate-forming group,a carbamate-forming group, a urea-forming group, an amino acidconjugate, and a polypeptide conjugate.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of HCV infection inan infected animal comprising administering to said animal, apharmaceutical composition or formulation comprising an effective amountof a conjugate of formula II, or a pharmaceutically acceptable salt orsolvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of HCV infection inan infected animal comprising administering to said animal apharmaceutical composition or formulation comprising a conjugate offormula II, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of HCV infection inan infected animal comprising administering said animal with apharmaceutical combination composition or formulation comprising aneffective amount of a conjugate of formula II, or a pharmaceuticallyacceptable salt or solvate thereof, and a second compound havinganti-HCV properties.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of formula II,or a pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable excipient.

In one embodiment, the invention also provides a method of inhibiting aviral enzyme comprising the step of contacting a sample suspected ofcontaining viral infected cells or tissues with a conjugate of formulaII, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of a viral infectionin an animal which comprises administering to said animal a formulationcomprising a therapeutically effective amount of a conjugate of formulaII, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides the use of a conjugate offormula II, or a pharmaceutically acceptable salt or solvate thereof toprepare a medicament for treatment of HCV.

In one embodiment, the invention also provides a conjugate of formulaII, or a pharmaceutically acceptable salt or solvate thereof, which iscapable of accumulating in human PBMC.

In one embodiment, the invention also provides a conjugate wherein thebioavailability of the conjugate or an intracellular metabolite of theconjugate in human PBMC is improved compared to the corresponding analoglacking the phosphonate group. For example, in one embodiment, thehalf-life is improved by at least about 50%; in another embodiment, thehalf-life is improved by at least about 100%; and in another embodiment,the half-life is improved by greater than 100%.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of formula II,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an AIDS treatment agent selected from an HIV inhibitor agent,an anti-infective agent, and an immunomodulator.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula II,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an HIV-protease inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula II,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of a reverse transcriptase inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula II,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of a non-nucleoside reverse transcriptase inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of Formula II,or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an HIV integrase inhibitor.

In one embodiment, the invention also provides a process for making apharmaceutical composition comprising combining a conjugate of formulaII, or a pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable excipient.

In one embodiment, the invention also provides a method of inhibitingRNA-dependent RNA polymerase comprising administering to a mammal inneed of such treatment, a therapeutically effective amount of aconjugate of Formula II, or a pharmaceutically acceptable salt orsolvate thereof.

In one embodiment, the invention also provides a method of treating anHCV infection comprising administering to a mammal in need of suchtreatment a therapeutically effective amount of a conjugate of formulaII, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method of treating adisorder affecting white blood cells comprising: administering aconjugate of Formula II, or a pharmaceutically acceptable salt orsolvate thereof to a patient in need of white-blood-cell targeting.

In one embodiment, the invention also provides a method of manufacturingan HCV inhibitor conjugate having both selectivity for white blood cellsand a desired pharmaceutical activity, comprising: chemicallysynthesizing a conjugate of Formula II (as described herein), whereinsaid conjugate differs from a second structure of a compound known tohave said desired pharmaceutical activity by having at least onehydrogen atom of said second structure replaced by an organicsubstituent comprising a prodrug moiety or incipient prodrug moiety.

In one embodiment, the invention also provides a method of accumulatingan RNA-dependent RNA polymerase inhibitor compound inside a white bloodcell, comprising administering to a sample, a composition comprising aconjugate of formula II, or a pharmaceutically acceptable salt orsolvate thereof. In one specific embodiment, said sample is a patient.

Conjugates of Formula III

In one embodiment, the invention provides a conjugate of Formula III:

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

B is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-D]pyrimidine;

X is selected from O, C(R^(y))₂, OC(R^(y))₂, NR and S;

Z is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F, Cl,Br, and I;

Y¹ is independently O, S, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), or N—NR₂;

Y² is independently O, CR₂, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S,S—S, S(O), or S(O)₂;

M2 is 0, 1 or 2;

R^(y) is independently H, F, Cl, Br, I, OH, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₉ alkyl, C₁-C₈ alkalihalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, alkylsulfone (—SO₂R), arylsulfone(—SO₂Ar), arylsulfoxide (—SOAr), arylthio (—SAr), sulfonamide (—SO₂NR₂),alkylsulfoxide (—SOR), ester (—C(═O)OR), amido (—C(═O)NR₂), 5-7 memberedring lactam, 5-7 membered ring lactone, nitrile (—CN), azido (—N₃),nitro (—NO₂), C₁-C₈ alkoxy (—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl,C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, polyethyleneoxy, or W³; orwhen taken together, R^(y) forms a carbocyclic ring of 3 to 7 carbonatoms;

R^(x) is independently R^(y), a protecting group, or the formula:

wherein:

M1a, M1c, and M1d are independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and

R is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₈substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, or a protecting group; and

W³ is W⁴ or W⁵, where W⁴ is R, —C(Y¹)R^(y), —C(Y¹)W⁵, —SO₂R^(y), or—SO₂W⁵; and W⁵ is a carbocycle or a heterocycle wherein W⁵ isindependently substituted with 0 to 3 R^(y) groups.

For a conjugate of Formula III, in one specific embodiment, C₁-C₈substituted alkyl, C₁-C₈ substituted alkenyl, C₁-C₈ substituted alkynyl,C₆-C₂₀ substituted aryl, and C₂-C₂₀ substituted heterocycle areindependently substituted with one or more substituents selected from F,Cl, Br, I, OH, —NH₂, —NH₃ ⁺, —NHR, —NR₂, —NR₃ ⁺, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₉ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, —SO₂R, —SO₂Ar, —SOAr, —SAr, —SO₂NR₂,—SOR, —CO₂R, —C(═O)NR₂, 5-7 membered ring lactam, 5-7 membered ringlactone, —CN, —N₃, —NO₂, C₁-C₉ alkoxy, C₁-C₉ trifluoroalkyl, C₁-C₈alkyl, C₃-C₁₂ carbocycle, C₆-C₂₀ aryl, C₂-C₂₀ heterocycle,polyethyleneoxy, phosphonate, phosphate, and a prodrug moiety.

For a conjugate of Formula III, in one specific embodiment, “protectinggroup” is selected from a carboxyl ester, a carboxamide, an aryl ether,an alkyl ether, a trialkylsilyl ether, a sulfonic acid ester, acarbonate, and a carbamate.

In one specific embodiment, for a conjugate of Formula III, W⁵ isselected from the structures:

In one specific embodiment, for a conjugate of Formula III, X is O andeach R^(y) is H.

In one specific embodiment, the conjugate of Formula III is a resolvedenantiomer having the structure:

In one specific embodiment, the conjugate of Formula III is a resolvedenantiomer having the structure:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

wherein R² is H or C₁-C₈ alkyl.

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

wherein in a more specific embodiment, Z is H and B is adenine.

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

wherein Y^(2c) is O, N(R^(y)) or S.

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

wherein, in a more specific embodiment, Y^(2c) is O or N(CH₃).

In one specific embodiment, for a conjugate of Formula III, substitutedtriazole has the structure:

In one specific embodiment, the conjugate of Formula III has thefollowing formula:

wherein:

B is selected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-D]pyrimidine;

X is selected from O, C(R^(y))₂, OC(R^(y))₂, NR and S;

Z is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR, F, Cl,Br, and I;

Y² is independently O, CR₂, NR, ⁺N(O)(R), N(OR), ⁺N(O)(OR), N—NR₂, S,S—S, S(O), or S(O)₂;

R^(y) is independently H, F, Cl, Br, I, OH, —C(═Y¹)R, —C(═Y¹)OR,—C(═Y¹)N(R)₂, —N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR),—S(O)₂(OR), —OC(═Y¹)R, —OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R,—SC(═Y¹)OR, —SC(═Y¹)(N(R)₂), —N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or—N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium (—NH₃ ⁺), alkylamino,dialkylamino, trialkylammonium, C₁-C₈ alkyl, C₁-C₈ alkylhalide,carboxylate, sulfate, sulfamate, sulfonate, 5-7 membered ring sultam,C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino, 4-dialkylaminopyridinium, C₁-C₈alkylhydroxyl, C₁-C₈ alkylthiol, alkylsulfone (—SO₂R), arylsulfone(—SO₂Ar), arylsulfoxide (—SOAr), arylthio (—SAr), sulfonamide (—SO₂NR₂),alkylsulfoxide (—SOR), ester (—C(═O)OR), amido (—C(═O)NR₂), 5-7 memberedring lactam, 5-7 membered ring lactone, nitrile (—CN), azido (—N₃),nitro (—NO₂), C₁-C₈ alkoxy (—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl,C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, polyethyleneoxy, or W³; orwhen taken together, R^(y) forms a carbocyclic ring of 3 to 7 carbonatoms;

R is C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₈substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, or a protecting group; and

PG is a protecting group selected from an ether-forming group, anester-forming group, a silyl-ether forming group, an amide-forminggroup, an acetal-forming group, a ketal-forming group, acarbonate-forming group, a carbamate-forming group, an amino acid, and apolypeptide.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of viral infection inan infected animal comprising administering to said animal, apharmaceutical composition or formulation comprising an effective amountof a conjugate of formula III, or a pharmaceutically acceptable salt orsolvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of viral infection inan infected animal comprising administering to said animal apharmaceutical composition or formulation comprising a conjugate offormula III, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of viral infection inan infected animal comprising administering said animal with apharmaceutical combination composition or formulation comprising aneffective amount of a conjugate of formula III, or a pharmaceuticallyacceptable salt or solvate thereof, and a second compound havingantiviral properties.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of formulaIII, or a pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable excipient.

In one embodiment, the invention also provides a method of inhibiting aviral enzyme comprising the step of contacting a sample suspected ofcontaining viral infected cells or tissues with a conjugate of formulaIII, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method for thetreatment or prevention of the symptoms or effects of a viral infectionin an animal which comprises administering to said animal a formulationcomprising a therapeutically effective amount of a conjugate of formulaIII, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides the use of a conjugate offormula III, or a pharmaceutically acceptable salt or solvate thereof toprepare a medicament for treatment of viral infection.

In one embodiment, the invention also provides a conjugate of formulaIII, or a pharmaceutically acceptable salt or solvate thereof, which iscapable of accumulating in human PBMC.

In one embodiment, the invention also provides a conjugate (e.g. offormula III) wherein the bioavailability of the conjugate or anintracellular metabolite of the conjugate in human PBMC is improvedcompared to the corresponding analog lacking the phosphonate group. Forexample, in one embodiment, the half-life is improved by at least about50%; in another embodiment, the half-life is improved by at least about100%; and in another embodiment, the half-life is improved by greaterthan 100%.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of FormulaIII, or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an AIDS treatment agent selected from an HIV inhibitor agent,an anti-infective agent, and an immunomodulator.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of FormulaIII, or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an HIV-protease inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of FormulaIII, or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of a reverse transcriptase inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of FormulaIII, or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of a non-nucleoside reverse transcriptase inhibitor.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising an effective amount of a conjugate of FormulaIII, or a pharmaceutically acceptable salt or solvate thereof; apharmaceutically acceptable excipient; and a therapeutically effectiveamount of an HIV integrase inhibitor.

In one embodiment, the invention also provides a process for making apharmaceutical composition comprising combining a conjugate of formulaIII, or a pharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable excipient.

In one embodiment, the invention also provides a method of inhibitingRNA-dependent RNA polymerase comprising administering to a mammal inneed of such treatment, a therapeutically effective amount of aconjugate of Formula III, or a pharmaceutically acceptable salt orsolvate thereof.

In one embodiment, the invention also provides a method of treating anHCV infection comprising administering to a mammal in need of suchtreatment a therapeutically effective amount of a conjugate of formulaIII, or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the invention also provides a method of treating adisorder affecting white blood cells comprising: administering aconjugate of Formula III, or a pharmaceutically acceptable salt orsolvate thereof to a patient in need of white-blood-cell targeting.

In one embodiment, the invention also provides a method of manufacturingan HCV inhibitor conjugate having both selectivity for white blood cellsand a desired pharmaceutical activity, comprising: chemicallysynthesizing a conjugate of Formula III (as described herein), whereinsaid conjugate differs from a second structure of a compound known tohave said desired pharmaceutical activity by having at least onehydrogen atom of said second structure replaced by an organicsubstituent comprising a prodrug moiety or incipient prodrug moiety.

In one embodiment, the invention also provides a method of accumulatingan RNA-dependent RNA polymerase inhibitor compound inside a white bloodcell, comprising administering to a sample, a composition comprising aconjugate of formula III, or a pharmaceutically acceptable salt orsolvate thereof. In one specific embodiment, said sample is a patient.

Linking Groups and Linkers

The invention provides conjugates that comprise an antiviral compoundthat is linked to one or more phosphonate groups either directly (e.g.through a covalent bond) or through a linking group (i.e. a linker) Thenature of the linker is not critical provided it does not interfere withthe ability of the phosphonate containing compound to function as atherapeutic agent. The phosphonate or the linker can be linked to thecompound (e.g. a compound of Formula 501-569) at any syntheticallyfeasible position on the compound by removing a hydrogen or any portionof the compound to provide an open valence for attachment of thephosphonate or the linker.

In one embodiment of the invention the linking group or linker (whichcan be designated “L”) can include all or a portions of the group A⁰,A¹, A², or W³ described herein.

In another embodiment of the invention the linking group or linker has amolecular weight of from about 20 daltons to about 400 daltons.

In another embodiment of the invention the linking group or linker has alength of about 5 angstroms to about 300 angstroms.

In another embodiment of the invention the linking group or linkerseparates the DRUG and a P(═Y¹) residue by about 5 angstroms to about200 angstroms, inclusive, in length.

In another embodiment of the invention the linking group or linker is adivalent, branched or unbranched, saturated or unsaturated, hydrocarbonchain, having from 2 to 25 carbon atoms, wherein one or more (e.g. 1, 2,3, or 4) of the carbon atoms is optionally replaced by (—O—), andwherein the chain is optionally substituted on carbon with one or more(e.g. 1, 2, 3, or 4) substituents selected from (C₁-C₆)alkoxy,(C₃-C₆)cycloalkyl, (C₁-C₆)alkanoyl, (C₁-C₆)alkanoyloxy,(C₁-C₆)alkoxycarbonyl, (C₁-C₆)alkylthio, azido, cyano, nitro, halo,hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, andheteroaryloxy.

In another embodiment of the invention the linking group or linker is ofthe formula W-A wherein A is (C₁-C₂₄)alkyl, (C₂-C₂₄)alkenyl,(C₂-C₂₄)alkynyl, (C₃-C₈)cycloalkyl, (C₆-C₁₀)aryl or a combinationthereof, wherein W is —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—,—S—, —S(O)—, —S(O)₂—, —N(R)—, —C(═O)—, or a direct bond; wherein each Ris independently H or (C₁-C₆)alkyl.

In another embodiment of the invention the linking group or linker is adivalent radical formed from a peptide.

In another embodiment of the invention the linking group or linker is adivalent radical formed from an amino acid.

In another embodiment of the invention the linking group or linker is adivalent radical formed from poly-L-glutamic acid, poly-L-aspartic acid,poly-L-histidine, poly-L-ornithine, poly-L-serine, poly-L-threonine,poly-L-tyrosine, poly-L-leucine, poly-L-lysine-L-phenylalanine,poly-L-lysine or poly-L-lysine-L-tyrosine.

In another embodiment of the invention the linking group or linker is ofthe formula W—(CH₂)_(n) wherein, n is between about 1 and about 10; andW is —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—,—S(O)₂—, —C(═O)—, —N(R)—, or a direct bond; wherein each R isindependently H or (C₁-C₆)alkyl.

In another embodiment of the invention the linking group or linker ismethylene, ethylene, or propylene.

In another embodiment of the invention the linking group or linker isattached to the phosphonate group through a carbon atom of the linker.

Intracellular Targeting

The phosphonate group of the compounds of the invention may cleave invivo in stages after they have reached the desired site of action, i.e.inside a cell. One mechanism of action inside a cell may entail a firstcleavage, e.g. by esterase, to provide a negatively-charged “locked-in”intermediate. Cleavage of a terminal ester grouping in a compound of theinvention thus affords an unstable intermediate which releases anegatively charged “locked in” intermediate.

After passage inside a cell, intracellular enzymatic cleavage ormodification of the phosphonate or prodrug compound may result in anintracellular accumulation of the cleaved or modified compound by a“trapping” mechanism. The cleaved or modified compound may then be“locked-in” the cell by a significant change in charge, polarity, orother physical property change which decreases the rate at which thecleaved or modified compound can exit the cell, relative to the rate atwhich it entered as the phosphonate prodrug. Other mechanisms by which atherapeutic effect are achieved may be operative as well. Enzymes whichare capable of an enzymatic activation mechanism with the phosphonateprodrug compounds of the invention include, but are not limited to,amidases, esterases, microbial enzymes, phospholipases, cholinesterases,and phosphatases.

In selected instances in which the drug is of the nucleoside type, suchas is the case of zidovudine and numerous other antiretroviral agents,it is known that the drug is activated in vivo by phosphorylation. Suchactivation may occur in the present system by enzymatic conversion ofthe “locked-in” intermediate with phosphokinase to the activephosphonate diphosphate and/or by phosphorylation of the drug itselfafter its release from the “locked-in” intermediate as described above.In either case, the original nucleoside-type drug will be convened, viathe derivatives of this invention, to the active phosphorylated species.

From the foregoing, it will be apparent that many different drugs can bederivatized in accord with the present invention. Numerous such drugsare specifically mentioned herein. However, it should be understood thatthe discussion of drug families and their specific members forderivatization according to this invention is not intended to beexhaustive, but merely illustrative.

Antiviral Compounds

The compounds of the invention include those with antiviral activity.The compounds of the inventions bear one or more (e.g., 1, 2, 3, or 4)phosphonate groups, which may be a prodrug moiety.

The term “antiviral compound” includes those compounds with antiviralactivity. In particular, the compounds include Dehydroepiandrosterone,LY-582563, L-Fd4C, L-FddC, telbivudine, clevudine, macrocyclic proteaseinhibitors, dOTCP, dOTC, DDL DDLP, ddcP, ddC, DADP, DAPD, d4TP, D4T,3TC, 3TCP FTCP, ABCP, AZT, IsoddAP, FTC, HCV polymerase inhibitors,ribavirin, viramidine, L-enantiomers of rib avirin and viramidine,levovirin, alkovirs, imiquimod, resquimod,4-(3-benzyl-phenyl)-2-hydroxy-4-oxo-but-2-enoic acid, propenonederivatives having HIV inhibiting activities, aza, polyazanaphthalenylcarboxamides, betulinic acid, dihydrobetulinic acid, isodd a, UT-231B,VX-148, gemcitabine, merimepodib, levamisole, mycophenolate, entecavir,foscamet, carbovir, abacavir, and BCX-1777.

Typically, compounds of the invention have a molecular weight of fromabout 400 amu to about 10,000 amu; in a specific embodiment of theinvention, compounds have a molecular weight of less than about 5000amu; in another specific embodiment of the invention, compounds have amolecular weight of less than about 2500 amu; in another specificembodiment of the invention, compounds have a molecular weight of lessthan about 1000 amu; in another specific embodiment of the invention,compounds have a molecular weight of less than about 800 amu; in anotherspecific embodiment of the invention, compounds have a molecular weightof less than about 600 amu; and in another specific embodiment of theinvention, compounds have a molecular weight of less than about 600 amuand a molecular weight of greater than about 400 amu.

The compounds of the invention also typically have a log D (polarity)less than about 5. In one embodiment the invention provides compoundshaving a log D less than about 4; in another one embodiment theinvention provides compounds having a log D less than about 3; inanother one embodiment the invention provides compounds having a log Dgreater than about −5; in another one embodiment the invention providescompounds having a log D greater than about −3; and in another oneembodiment the invention provides compounds having a log D greater thanabout 0 and less than about 3.

In one specific embodiment the invention provides compounds that mayfall within the generic definition of the term antiviral compound butwhich further comprise a phosphonate group, e.g., a phosphonate diester,phosphonamidate-ester prodrug, or a phosphondiamidate-ester (Jiang etal., US 2002/0173490 A1).

Selected substituents within the compounds of the invention are presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number maybe present in any given claim. For example, R^(x) contains a R¹substituent. R^(y) can be R², which in turn can be R³. If R³ is selectedto be R^(3c), then a second instance of R^(x) can be selected. One ofordinary skill in the art of medicinal chemistry understands that thetotal number of such substituents is reasonably limited by the desiredproperties of the compound intended. Such properties include, by ofexample and not limitation, physical properties such as molecularweight, solubility or log P, application properties such as activityagainst the intended target, and practical properties such as ease ofsynthesis.

By way of example and not limitation, W³, R^(y) and R³ are all recursivesubstituents in certain claims. Typically, each of these mayindependently occur 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1, or 0, times in a given claim. More typically, each ofthese may independently occur 12 or fewer times in a given claim. Moretypically yet, W³ will occur 0 to 8 times, R^(y) will occur 0 to 6 timesand R³ will occur 0 to 10 times in a given claim. Even more typically,W³ will occur 0 to 6 times, R^(y) will occur 0 to 4 times and R³ willoccur 0 to 8 times in a given claim.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal chemistry understands theversatility of such substituents. To the degree that recursivesubstituents are present in an claim of the invention, the total numberwill be determined as set forth above.

Whenever a compound described herein is substituted with more than oneof the same designated group, e.g., “R¹” or “R^(6a)”, then it will beunderstood that the groups may be the same or different, i.e., eachgroup is independently selected. Wavy lines indicate the site ofcovalent bond attachments to the adjoining groups, moieties, or atoms.

The phosphonate group may be a phosphonate prodrug moiety. The prodrugmoiety may be sensitive to hydrolysis, such as, but not limited to, apivaloyloxymethyl carbonate (POC) or POM group. Alternatively, theprodrug moiety may be sensitive to enzymatic potentiated cleavage, suchas a lactate ester or a phosphonamidate-ester group.

In one embodiment of the invention the compound is not ananti-inflammatory compound; in another embodiment the compound is not ananti-infective; in another embodiment the compound is not a compoundthat is active against immune-mediated conditions; in another embodimentthe compound is not an anti-cancer compound; in another embodiment thecompound is not a compound that is active against metabolic diseases; inanother embodiment the compound is not a nucleoside; in anotherembodiment the compound is not a IMPDH inhibitor; in another embodimentthe compound is not an antimetabolite; in another embodiment thecompound is not a PNP inhibitor; in another embodiment the compound isnot a substituted compound of any one of formulae 509-510, 556-557, and559-562; and in another embodiment the compound is not a compound of anyone of formulae 13-18, 72, 77-83, and 90-102.

In one embodiment of the invention, the compound is in an isolated andpurified form. Generally, the term “isolated and purified” means thatthe compound is substantially free from biological materials (e.g.blood, tissue, cells, etc.). In one specific embodiment of theinvention, the term means that the compound or conjugate of theinvention is at least about 50 wt. % free from biological materials; inanother specific embodiment, the term means that the compound orconjugate of the invention is at least about 75 wt. % free frombiological materials; in another specific embodiment, the term meansthat the compound or conjugate of the invention is at least about 90 wt.% free from biological materials; in another specific embodiment, theterm means that the compound or conjugate of the invention is at leastabout 98 wt. % free from biological materials; and in anotherembodiment, the term means that the compound or conjugate of theinvention is at least about 99 wt. % free from biological materials. Inanother specific embodiment, the invention provides a compound orconjugate of the invention that has been synthetically prepared (e.g.,ex vivo).

Cellular Accumulation

In one embodiment, the invention is provides compounds capable ofaccumulating in human PBMC (peripheral blood mononuclear cells). PBMCrefer to blood cells having round lymphocytes and monocytes.Physiologically, PBMC are critical components of the mechanism againstinfection. PBMC may be isolated from heparinized whole blood of normalhealthy donors or buffy coats, by standard density gradientcentrifugation and harvested from the interface, washed (e.g.phosphate-buffered saline) and stored in freezing medium. PBMC may becultured in multi-well plates. At various times of culture, supernatantmay be either removed for assessment, or cells may be harvested andanalyzed (Smith R. et al (2003) Blood 102(7):2532-2540). The compoundsof this claim may further comprise a phosphonate or phosphonate prodrug.More typically, the phosphonate or phosphonate prodrug can have thestructure A³ as described herein.

Typically, compounds of the invention demonstrate improved intracellularhalf-life of the compounds or intracellular metabolites of the compoundsin human PBMC when compared to analogs of the compounds not having thephosphonate or phosphonate prodrug. Typically, the half-life is improvedby at least about 50%, more typically at least in the range 50-100%,still more typically at least about 100%, more typically yet greaterthan about 100%.

In one embodiment of the invention the intracellular half-life of ametabolite of the compound in human PBMCs is improved when compared toan analog of the compound not having the phosphonate or phosphonateprodrug. In such claims, the metabolite may be generatedintracellularly, e.g. generated within human PBMC. The metabolite may bea product of the cleavage of a phosphonate prodrug within human PBMCs.The phosphonate prodrug may be cleaved to form a metabolite having atleast one negative charge at physiological pH. The phosphonate prodrugmay be enzymatically cleaved within human PBMC to form a phosphonatehaving at least one active hydrogen atom of the form P—OH.

Stereoisomers

The compounds of the invention may have chiral centers, e.g., chiralcarbon or phosphorus atoms. The compounds of the invention thus includeracemic mixtures of all stereoisomers, including enantiomers,diastereomers, and atropisomers. In addition, the compounds of theinvention include enriched or resolved optical isomers at any or allasymmetric, chiral atoms. In other words, the chiral centers apparentfrom the depictions are provided as the chiral isomers or racemicmixtures. Both racemic and diastereomeric mixtures, as well as theindividual optical isomers isolated or synthesized, substantially freeof their enantiomeric or diastereomeric partners, are all within thescope of the invention. The racemic mixtures are separated into theirindividual, substantially optically pure isomers through well-knowntechniques such as, for example, the separation of diastereomeric saltsformed with optically active adjuncts, e.g., acids or bases followed byconversion back to the optically active substances. In most instances,the desired optical isomer is synthesized by means of stereospecificreactions, beginning with the appropriate stereoisomer of the desiredstarting material.

The compounds of the invention can also exist as tautomeric isomers incertain cases. All though only one delocalized resonance structure maybe depicted, all such forms are contemplated within the scope of theinvention. For example, ene-amine tautomers can exist for purine,pyrimidine, imidazole, guanidine, amidine, and tetrazole systems and alltheir possible tautomeric forms are within the scope of the invention.

Salts and Hydrates

The compositions of this invention optionally comprise salts of thecompounds herein, especially pharmaceutically acceptable non-toxic saltscontaining, for example, Na⁺, Li⁺, K⁺, Ca⁺² and Mg⁺². Such salts mayinclude those derived by combination of appropriate cations such asalkali and alkaline earth metal ions or ammonium and quaternary aminoions with an acid anion moiety, typically a carboxylic acid. Monovalentsalts are preferred if a water soluble salt is desired.

Metal salts typically are prepared by reacting the metal hydroxide witha compound of this invention. Examples of metal salts which are preparedin this way are salts containing Li⁺, Na⁺, and K⁺. A less soluble metalsalt can be precipitated from the solution of a more soluble salt byaddition of the suitable metal compound.

In addition, salts may be formed from acid addition of certain organicand inorganic acids, e.g., HCl, HBr, H₂SO₄, H₃PO₄ or organic sulfonicacids, to basic centers, typically amines, or to acidic groups. Finally,it is to be understood that the compositions herein comprise compoundsof the invention in their un-ionized, as well as zwitterionic form, andcombinations with stoichiometric amounts of water as in hydrates.

Also included within the scope of this invention are the salts of theparental compounds with one or more amino acids. Any of the amino acidsdescribed above are suitable, especially the naturally-occurring aminoacids found as protein components, although the amino acid typically isone bearing a side chain with a basic or acidic group, e.g., lysine,arginine or glutamic acid, or a neutral group such as glycine, serine,threonine, alanine, isoleucine, or leucine.

Methods of Inhibition of Viral Infections

Another aspect of the invention relates to methods of inhibiting viralinfections, comprising the step of treating a sample or subjectsuspected of needing such inhibition with a composition of theinvention.

Compositions of the invention may act as inhibitors of viral infections,or as intermediates for such inhibitors or have other utilities asdescribed below. The inhibitors will bind to locations on the surface orin a cavity of a cell having a unique geometry. Compositions binding acell may bind with varying degrees of reversibility. Those compoundsbinding substantially irreversibly are ideal candidates for use in thismethod of the invention. Once labeled, the substantially irreversiblybinding compositions are useful as probes for the detection of viruses.Accordingly, the invention relates to methods of detecting viruses in asample or subject suspected of containing a virus, comprising the stepsof: treating such a sample or subject with a composition comprising acompound of the invention bound to a label; and observing the effect ofthe sample on the activity of the label. Suitable labels are well knownin the diagnostics field and include stable free radicals, fluorophores,radioisotopes, enzymes, chemiluminescent groups and chromogens. Thecompounds herein are labeled in conventional fashion using functionalgroups such as hydroxyl or amino.

Within the context of the invention samples suspected of containing avirus include natural or man-made materials such as living organisms;tissue or cell cultures; biological samples such as biological materialsamples (blood, serum, urine, cerebrospinal fluid, tears, sputum,saliva, tissue samples, and the like); laboratory samples; food, water,or air samples; bioproduct samples such as extracts of cells,particularly recombinant cells synthesizing a desired glycoprotein; andthe like. Typically the sample will be suspected of containing anorganism which induces a viral infection, frequently a pathogenicorganism such as an tumor virus. Samples can be contained in any mediumincluding water and organic solvent\water mixtures. Samples includeliving organisms such as humans, and man made materials such as cellcultures.

The treating step of the invention comprises adding the composition ofthe invention to the sample or it comprises adding a precursor of thecomposition to the sample. The addition step comprises any method ofadministration as described above.

If desired, the anti-virus activity of a compound of the invention afterapplication of the composition can be observed by any method includingdirect and indirect methods of detecting such activity. Quantitative,qualitative, and semiquantitative methods of determining such activityare all contemplated. Typically one of the screening methods describedabove are applied, however, any other method such as observation of thephysiological properties of a living organism are also applicable.

Screens for Antiviral Compounds

Compositions of the invention are screened for antiviral activity by anyof the conventional techniques for evaluating enzyme activity. Withinthe context of the invention, typically compositions are first screenedfor inhibitory activity in vitro and compositions showing inhibitoryactivity are then screened for activity in vivo. Compositions having invitro Ki (inhibitory constants) of less then about 5×10⁻⁶ M, typicallyless than about 1×10⁻⁷ M and preferably less than about 5×10⁻⁸ M arepreferred for in vivo use.

Useful in vitro screens have been described in detail and will not beelaborated here.

Pharmaceutical Formulations

The compounds of this invention are formulated with conventionalcarriers and excipients, which will be selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, bindersand the like. Aqueous formulations are prepared in sterile form, andwhen intended for delivery by other than oral administration generallywill be isotonic. All formulations will optionally contain excipientssuch as those set forth in the Handbook of Pharmaceutical Excipients(1986). Excipients include ascorbic acid and other antioxidants,chelating agents such as EDTA, carbohydrates such as dextrin,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and thelike. The pH of the formulations ranges from about 3 to about 11, but isordinarily about 7 to 10.

While it is possible for the active ingredients to be administered aloneit may be preferable to present them as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the inventioncomprise at least one active ingredient, as above defined, together withone or more acceptable carriers therefor and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand physiologically innocuous to the recipient thereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

For administration to the eye or other external tissues e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w (including active ingredient(s) in a range between 0.1%and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. The topical formulations maydesirably include a compound which enhances absorption or penetration ofthe active ingredient through the skin or other affected areas. Examplesof such dermal penetration enhancers include dimethyl sulphoxide andrelated analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. The cream should preferablybe a non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils are used.

Pharmaceutical formulations according to the present invention compriseone or more compounds of the invention together with one or morepharmaceutically acceptable carriers or excipients and optionally othertherapeutic agents. Pharmaceutical formulations containing the activeingredient may be in any form suitable for the intended method ofadministration. When used for oral use for example, tablets, troches,lozenges, aqueous or oil suspensions, dispersible powders or granules,emulsions, hard or soft capsules, syrups or elixirs may be prepared.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsincluding sweetening agents, flavoring agents, coloring agents andpreserving agents, in order to provide a palatable preparation. Tabletscontaining the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, lactosemonohydrate, croscarmellose sodium, povidone, calcium or sodiumphosphate; granulating and disintegrating agents, such as maize starch,or alginic acid; binding agents, such as cellulose, microcrystallinecellulose, starch, gelatin or acacia; and lubricating agents, such asmagnesium stearate, stearic acid or talc. Tablets may be uncoated or maybe coated by known techniques including microencapsulation to delaydisintegration and adsorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearatealone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water providethe active ingredient in admixture with a dispersing or wetting agent, asuspending agent, and one or more preservatives. Suitable dispersing orwetting agents and suspending agents are exemplified by those disclosedabove. Additional excipients, for example sweetening, flavoring andcoloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate. Theemulsion may also contain sweetening and flavoring agents. Syrups andelixirs may be formulated with sweetening agents, such as glycerol,sorbitol or sucrose. Such formulations may also contain a demulcent, apreservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for administration to the eye include eye dropswherein the active ingredient is dissolved or suspended in a suitablecarrier, especially an aqueous solvent for the active ingredient. Theactive ingredient is preferably present in such formulations in aconcentration of 0.5 to 20%, advantageously 0.5 to 10% particularlyabout 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of viral infections as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Compounds of the invention can also be formulated to provide controlledrelease of the active ingredient to allow less frequent dosing or toimprove the pharmacokinetic or toxicity profile of the activeingredient. Accordingly, the invention also provided compositionscomprising one or more compounds of the invention formulated forsustained or controlled release.

Effective dose of active ingredient depends at least on the nature ofthe condition being treated, toxicity, whether the compound is beingused prophylactically (lower doses) or against an active viralinfection, the method of delivery, and the pharmaceutical formulation,and will be determined by the clinician using conventional doseescalation studies. It can be expected to be from about 0.0001 to about100 mg/kg body weight per day. Typically, from about 0.01 to about 10mg/kg body weight per day. More typically, from about 0.01 to about 5mg/kg body weight per day. More typically, from about 0.05 to about 0.5mg/kg body weight per day. For example, the daily candidate dose for anadult human of approximately 70 kg body weight will range from 1 mg to1000 mg, preferably between 5 mg and 500 mg, and may take the form ofsingle or multiple doses.

Routes of Administration

One or more compounds of the invention (herein referred to as the activeingredients) are administered by any route appropriate to the conditionto be treated. Suitable routes include oral, rectal, nasal, topical(including buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural), and the like. It will be appreciated that the preferred routemay vary with for example the condition of the recipient. An advantageof the compounds of this invention is that they are orally bioavailableand can be dosed orally.

Combination Therapy

Active ingredients of the invention are also used in combination withother active ingredients. Such combinations are selected based on thecondition to be treated, cross-reactivities of ingredients andpharmaco-properties of the combination. For example, when treating aviral infection the compositions of the invention can be combined withother agents that are effective to treat a viral infection (such asother antiviral agents).

It is also possible to combine any compound of the invention with one ormore other active ingredients in a unitary dosage form for simultaneousor sequential administration to a patient. The combination therapy maybe administered as a simultaneous or sequential regimen. Whenadministered sequentially, the combination may be administered in two ormore administrations.

The combination therapy may provide “synergy” and “synergistic effect”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined formulation; (2) delivered by alternationor in parallel as separate formulations; or (3) by some other regimen.When delivered in alternation therapy, a synergistic effect may beattained when the compounds are administered or delivered sequentially,e.g., in separate tablets, pills or capsules, or by different injectionsin separate syringes. In general, during alternation therapy, aneffective dosage of each active ingredient is administered sequentially,i.e. serially, whereas in combination therapy, effective dosages of twoor more active ingredients are administered together.

Metabolites of the Compounds of the Invention

Also falling within the scope of this invention are the in vivometabolic products of the compounds described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,esterification and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof. Such products typically are identified by preparing aradiolabelled (e.g., C¹⁴ or H³) compound of the invention, administeringit parenterally in a detectable dose (e.g., greater than about 0.5mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man,allowing sufficient time for metabolism to occur (typically about 30seconds to 30 hours) and isolating its conversion products from theurine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS or NMR analysis. In general, analysis of metabolites is done in thesame way as conventional drug metabolism studies well-known to thoseskilled in the art. The conversion products, so long as they are nototherwise found in vivo, are useful in diagnostic assays for therapeuticdosing of the compounds of the invention even if they possess noantiviral activity of their own.

Recipes and methods for determining stability of compounds in surrogategastrointestinal secretions are known. Compounds are defined herein asstable in the gastrointestinal tract where less than about 50 molepercent of the protected groups are deprotected in surrogate intestinalor gastric juice upon incubation for 1 hour at 37° C. Simply because thecompounds are stable to the gastrointestinal tract does not mean thatthey cannot be hydrolyzed in vivo. The phosphonate prodrugs of theinvention typically will be stable in the digestive system but aresubstantially hydrolyzed to the parental drug in the digestive lumen,liver or other metabolic organ, or within cells in general.

Antiviral Activity

The antiviral activity of a compound of the invention can be measuredusing standard screening protocols that are known. For example, theantiviral activity of a compound can be measured in a cell culture assayusing the following general protocol.

Antiviral Cell Culture Assay

The assay is based on quantification of the antiviral effect by acolorimetric detection of the viability of virus-infected cells in thepresence or absence of tested inhibitors. The compound-induced celldeath is determined using a metabolic substrate2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT) which is converted only by intact cells into a product withspecific absorption characteristics as described by Weislow O S, KiserR, Fine D L, Bader J, Shoemaker R H and Boyd M R (1989) J Natl CancerInst 81, 577.

Assay Protocol for Determination of EC50:

-   1. Maintain MT2 cells in RPMI-1640 medium supplemented with 5% fetal    bovine serum and antibiotics.-   2. Infect the cells with the viral agent for 3 hours at 37° C. using    the virus inoculum corresponding to a multiplicity of infection    equal to 0.01.-   3. Distribute the infected cells into a 96-well plate (20,000 cells    in 100 μl/well) and add various concentrations of the tested    inhibitor in triplicate (100 μl/well in culture media). Include    untreated infected and untreated mock-infected control cells.-   4. Incubate the cells for 5 days at 37° C.-   5. Prepare a compound solution (6 ml per assay plate) at a    concentration of 2 mg/ml in a phosphate-buffered saline pH 7.4. Heat    the solution in water-bath for 5 min at 55° C. Add 50 μl of    N-methylphenazonium methasulfate (5 μg/ml) per 6 ml of XTT solution.-   6. Remove 100 μl media from each well on the assay plate.-   7. Add 100 μl of the XTT substrate solution per well and incubate at    37° C. for 45 to 60 min in a CO₂ incubator.-   8. Add 20 μl of 2% Triton X-100 per well to inactivate the virus.-   9. Read the absorbance at 450 nm with subtracting off the background    absorbance at 650 nm.-   10. Plot the percentage absorbance relative to untreated control and    estimate the EC50 value as drug concentration resulting in a 50%    protection of the infected cells.

The cytotoxicity of a compound of the invention can be determined usingthe following general protocol.

Cytotoxicity Cell Culture Assay (Determination of CC50):

The assay is based on the evaluation of cytotoxic effect of testedcompounds using a metabolic substrate.

Assay Protocol for Determination of CC50:

-   1. Maintain MT-2 cells in RPMI-1640 medium supplemented with 5%    fetal bovine serum and antibiotics.-   2. Distribute the cells into a 96-well plate (20,000 cell in 100 μl    media per well) and add various concentrations of the tested    compound in triplicate (100 μl/well). Include untreated control.-   3. Incubate the cells for 5 days at 37° C.-   4. Prepare XTT solution (6 ml per assay plate) in dark at a    concentration of 2 mg/ml in a phosphate-buffered saline pH 7.4. Heat    the solution in a water-bath at 55° C. for 5 min. Add 50 μl of    N-methylphenazonium methasulfate (5 μg/ml) per 6 ml of XTT solution.-   5. Remove 100 μl media from each well on the assay plate and add 100    μl of the XTT substrate solution per well. Incubate at 37° C. for 45    to 60 min in a CO₂ incubator.-   6. Add 20 μl of 2% Triton X-100 per well to stop the metabolic    conversion of XTT.-   7. Read the absorbance at 450 nm with subtracting off the background    at 650 nm.-   8. Plot the percentage absorbance relative to untreated control and    estimate the CC50 value as drug concentration resulting in a 50%    inhibition of the cell growth. Consider the absorbance being    directly proportional to the cell growth.

Exemplary Methods of Making the Compounds of the Invention.

The invention also relates to methods of making the compositions of theinvention. The compositions are prepared by any of the applicabletechniques of organic synthesis. Many such techniques are well known inthe art. However, many of the known techniques are elaborated inCompendium of Organic Synthetic Methods (John Wiley & Sons, New York),Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, jr., 1980; Vol. 5, Leroy G. Wade,Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., AdvancedOrganic Chemistry, Third Edition, (John Wiley & Sons, New York, 1985),Comprehensive Organic Synthesis, Selectivity, Strategy & Efficiency inModern Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief(Pergamon Press, New York, 1993 printing).

A number of exemplary methods for the preparation of the compositions ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic, and reactiontimes will be 10 seconds to 10 days. Work-up typically consists ofquenching any unreacted reagents followed by partition between awater/organic layer system (extraction) and separating the layercontaining the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C., solvents are typically aprotic for reductions and may beeither protic or aprotic for oxidations. Reaction times are adjusted toachieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions).

Standard synthetic techniques such as azeotropic removal of reactionby-products and use of anhydrous reaction conditions (e.g., inert gasenvironments) are common in the art and will be applied when applicable.

Schemes and Examples

General aspects of these exemplary methods are described below and inthe Examples. Each of the products of the following processes isoptionally separated, isolated, and/or purified prior to its use insubsequent processes.

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic, and reactiontimes will be 10 seconds to 10 days. Work-up typically consists ofquenching any unreacted reagents followed by partition between awater/organic layer system (extraction) and separating the layercontaining the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C., solvents are typically aprotic for reductions and may beeither protic or aprotic for oxidations. Reaction times are adjusted toachieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions).

Standard synthetic techniques such as azeotropic removal of reactionby-products and use of anhydrous reaction conditions (e.g., inert gasenvironments) are common in the art and will be applied when applicable.

The terms “treated”, “treating”, “treatment”, and the like, when used inconnection with a chemical synthetic operation, mean contacting, mixing,reacting, allowing to react, bringing into contact, and other termscommon in the art for indicating that one or more chemical entities istreated in such a manner as to convert it to one or more other chemicalentities. This means that “treating compound one with compound two” issynonymous with “allowing compound one to react with compound two”,“contacting compound one with compound two”, “reacting compound one withcompound two”, and other expressions common in the art of organicsynthesis for reasonably indicating that compound one was “treated”,“reacted”, “allowed to react”, etc., with compound two. For example,treating indicates the reasonable and usual manner in which organicchemicals are allowed to react. Normal concentrations (0.01M to 10M,typically 0.1M to 1M), temperatures (−100° C. to 250° C., typically −78°C. to 150° C., more typically −78° C. to 100° C., still more typically0° C. to 100° C.), reaction vessels (typically glass, plastic, metal),solvents, pressures, atmospheres (typically air for oxygen and waterinsensitive reactions or nitrogen or argon for oxygen or watersensitive), etc., are intended unless otherwise indicated. The knowledgeof similar reactions known in the art of organic synthesis are used inselecting the conditions and apparatus for “treating” in a givenprocess. In particular, one of ordinary skill in the art of organicsynthesis selects conditions and apparatus reasonably expected tosuccessfully carry out the chemical reactions of the described processesbased on the knowledge in the art.

Modifications of each of the exemplary schemes and in the examples(hereafter “exemplary schemes”) leads to various analogs of the specificexemplary materials produce. The above-cited citations describingsuitable methods of organic synthesis are applicable to suchmodifications.

In each of the exemplary schemes it may be advantageous to separatereaction products from one another and/or from starting materials. Thedesired products of each step or series of steps is separated and/orpurified (hereinafter separated) to the desired degree of homogeneity bythe techniques common in the art. Typically such separations involvemultiphase extraction, crystallization from a solvent or solventmixture, distillation, sublimation, or chromatography. Chromatographycan involve any number of methods including, for example: reverse-phaseand normal phase; size exclusion; ion exchange; high, medium, and lowpressure liquid chromatography methods and apparatus; small scaleanalytical; simulated moving bed (SMB) and preparative thin or thicklayer chromatography, as well as techniques of small scale thin layerand flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point, and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

A single stereoisomer, e.g., an enantionier, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Stereochemistry of Carbon Compounds, (1962) by E. L.Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283-302). Racemic mixtures of chiral compounds of the invention can beseparated and isolated by any suitable method, including: (1) formationof ionic, diastereomeric salts with chiral compounds and separation byfractional crystallization or other methods, (2) formation ofdiastereomeric compounds with chiral derivatizing reagents, separationof the diastereomers, and conversion to the pure stereoisomers, and (3)separation of the substantially pure or enriched stereoisomers directlyunder chiral conditions.

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(Eliel, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds,John Wiley & Sons, Inc., p. 322). Diastereomeric compounds can be formedby reacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the free,enantiomerically enriched xanthene. A method of determining opticalpurity involves making chiral esters, such as a menthyl ester, e.g., (−)menthyl chloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. (1982) J. Org.Chem. 47:4165), of the racemic mixture, and analyzing the NMR spectrumfor the presence of the two atropisomeric diastereomers. Stablediastereomers of atropisomeric compounds can be separated and isolatedby normal- and reverse-phase chromatography following methods forseparation of atropisomeric naphthyl-isoquinolines (Hoye, T., WO96/15111). By method (3), a racemic mixture of two enantiomers can beseparated by chromatography using a chiral stationary phase (ChiralLiquid Chromatography (1989) W. J. Lough, Ed. Chapman and Hall, NewYork; Okamoto, (1990) J. of Chromatogr. 513:375-378). Enriched orpurified enantiomers can be distinguished by methods used to distinguishother chiral molecules with asymmetric carbon atoms, such as opticalrotation and circular dichroism.

EXAMPLES GENERAL SECTION

A number of exemplary methods for the preparation of compounds of theinvention are provided herein, for example, in the Examples hereinbelow.These methods are intended to illustrate the nature of such preparationsare not intended to limit the scope of applicable methods. Certaincompounds of the invention can be used as intermediates for thepreparation of other compounds of the invention. For example, theinterconversion of various phosphonate compounds of the invention isillustrated below.

Interconversions of the Phosphonates R-LINK-P(O)(OR¹)₂,R-LINK-P(O)(OR¹)(OH) AND R-LINK-P(O)(OH)₂.

The following schemes 32-38 described the preparation of phosphonateesters of the general structure R-link-P(O)(OR¹)₂, in which the groupsR¹ may be the same or different. The R¹ groups attached to a phosphonateester, or to precursors thereto, may be changed using establishedchemical transformations. The interconversion reactions of phosphonatesare illustrated in Scheme S32. The group R in Scheme 32 represents thesubstructure, i.e. the drug “scaffold, to which the substituentlink-P(O)(OR¹)₂ is attached, either in the compounds of the invention,or in precursors thereto. At the point in the synthetic route ofconducting a phosphonate interconversion, certain functional groups in Rmay be protected. The methods employed for a given phosphonatetransformation depend on the nature of the substituent R¹, and of thesubstrate to which the phosphonate group is attached. The preparationand hydrolysis of phosphonate esters is described in Organic PhosphorusCompounds, G. M. Kosolapoff, L. Maeir, eds, Wiley, 1976, p. 9ff.

In general, synthesis of phosphonate esters is achieved by coupling anucleophile amine or alcohol with the corresponding activatedphosphonate electrophilic precursor. For example, chlorophosphonateaddition on to 5′-hydroxy of nucleoside is a well known method forpreparation of nucleoside phosphate monoesters. The activated precursorcan be prepared by several well known methods. Chlorophosphonates usefulfor synthesis of the prodrugs are prepared from thesubstituted-1,3-propanediol (Wissner, et al, (1992) J. Med. Chem.35:1650). Chlorophosphonates are made by oxidation of the correspondingchlorophospholanes (Anderson, et al, (1984) J. Org. Chem. 49:1304) whichare obtained by reaction of the substituted diol with phosphorustrichloride. Alternatively, the chlorophosphonate agent is made bytreating substituted-1,3-diols with phosphorusoxychloride (Patois, etal, (1990) J. Chem. Soc. Perkin Trans. I, 1577). Chlorophosphonatespecies may also be generated in situ from corresponding cyclicphosphites (Silverburg, et al., (1996) Tetrahedron lett., 37:771-774),which in turn can be either made from chlorophospholane orphosphoramidate intermediate. Phosphoroflouridate intermediate preparedeither from pyrophosphate or phosphoric acid may also act as precursorin preparation of cyclic prodrugs (Watanabe et al., (1988) Tetrahedronlett., 29:5763-66).

Phosphonate prodrugs of the present invention may also be prepared fromthe free acid by Mitsunobu reactions (Mitsunobu, (1981) Synthesis, 1;Campbell, (1992) J. Org. Chem. 57:6331), and other acid couplingreagents including, but not limited to, carbodiimides (Alexander, et al,(1994) Collect. Czech. Chem. Commun. 59:1853; Casara et al, (1992)Bioorg. Med. Chem. Lett. 2:145; Ohashi et al, (1988) Tetrahedron Lett.,29:1189), and benzotriazolyloxytris-(dimethylamino)phosphonium salts(Campagne et al (1993) Tetrahedron Lett. 34:6743).

Aryl halides undergo Ni⁺² catalyzed reaction with phosphite derivativesto give aryl phosphonate containing compounds (Balthazar, et al (1980)J. Org. Chem. 45:5425). Phosphonates may also be prepared from thechlorophosphonate in the presence of a palladium catalyst using aromatictriflates (Petrakis et al (1987) J. Am. Chem. Soc. 109:2831; Lu et al(1987) Synthesis 726). In another method, aryl phosphonate esters areprepared from aryl phosphates under anionic rearrangement conditions(Melvin (1981) Tetrahedron Lett. 22:3375; Casteel et al (1991)Synthesis, 691). N-Alkoxy aryl salts with alkali metal derivatives ofcyclic alkyl phosphonate provide general synthesis forheteroaryl-2-phosphonate linkers (Redmore (1970) J. Org. Chem. 35:4114).These above mentioned methods can also be extended to compounds wherethe W⁵ group is a heterocycle. Cyclic-1,3-propanyl prodrugs ofphosphonates are also synthesized from phosphonic diacids andsubstituted propane-1,3-diols using a coupling reagent such as1,3-dicyclohexylcarbodiimide (DCC) in presence of a base (e.g.,pyridine). Other carbodiimide based coupling agents like1,3-disopropylcarbodiimide or water soluble reagent,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) canalso be utilized for the synthesis of cyclic phosphonate prodrugs.

The conversion of a phosphonate diester S32.1 into the correspondingphosphonate monoester S32.2 (Scheme 32, Reaction 1) is accomplished by anumber of methods. For example, the ester S32.1 in which R¹ is anaralkyl group such as benzyl, is converted into the monoester compoundS32.2 by reaction with a tertiary organic base such asdiazabicyclooctane (DABCO) or quinuclidine, as described in J. Org.Chem. (1995) 60:2946. The reaction is performed in an inert hydrocarbonsolvent such as toluene or xylene, at about 110° C. The conversion ofthe diester S32.1 in which R¹ is an aryl group such as phenyl, or analkenyl group such as allyl, into the monoester S32.2 is effected bytreatment of the ester S32.1 with a base such as aqueous sodiumhydroxide in acetonitrile or lithium hydroxide in aqueoustetrahydrofuran. Phosphonate diesters S32.1 in which one of the groupsR¹ is aralkyl, such as benzyl, and the other is alkyl, is converted intothe monoesters S32.2 in which R¹ is alkyl by hydrogenation, for exampleusing a palladium on carbon catalyst. Phosphonate diesters in which bothof the groups R¹ are alkenyl, such as allyl, is converted into themonoester S32.2 in which R¹ is alkenyl, by treatment withchlorotris(triphenylphosphine)rhodium (Wilkinson's catalyst) in aqueousethanol at reflux, optionally in the presence of diazabicyclooctane, forexample by using the procedure described in J. Org. Chem. (1973)38:3224, for the cleavage of allyl carboxylates.

The conversion of a phosphonate diester S32.1 or a phosphonate monoesterS32.2 into the corresponding phosphonic acid S32.3 (Scheme 32, Reactions2 and 3) can be effected by reaction of the diester or the monoesterwith trimethylsilyl bromide, as described in J. Chem. Soc., Chem. Comm.,(1979) 739. The reaction is conducted in an inert solvent such as, forexample, dichloromethane, optionally in the presence of a silylatingagent such as bis(trimethylsilyl)trifluoroacetamide, at ambienttemperature. A phosphonate monoester S32.2 in which R¹ is aralkyl suchas benzyl, is converted into the corresponding phosphonic acid S32.3 byhydrogenation over a palladium catalyst, or by treatment with hydrogenchloride in an ethereal solvent such as dioxane. A phosphonate monoesterS32.2 in which R¹ is alkenyl such as, for example, allyl, is convertedinto the phosphonic acid S32.3 by reaction with Wilkinson's catalyst inan aqueous organic solvent, for example in 15% aqueous acetonitrile, orin aqueous ethanol, for example using the procedure described in Helv.Chim. Acta. (1985) 68:618. Palladium catalyzed hydrogenolysis ofphosphonate esters S32.1 in which R¹ is benzyl is described in J. Org.Chem. (1959) 24:434. Platinum-catalyzed hydrogenolysis of phosphonateesters S32.1 in which R¹ is phenyl is described in J. Am. Chem. Soc.(1956) 78:2336.

The conversion of a phosphonate monoester S32.2 into a phosphonatediester S32.1 (Scheme 32, Reaction 4) in which the newly introduced R′group is alkyl, aralkyl, haloalkyl such as chloroethyl, or aralkyl iseffected by a number of reactions in which the substrate S32.2 isreacted with a hydroxy compound R¹OH, in the presence of a couplingagent. Typically, the second phosphonate ester group is different thanthe first introduced phosphonate ester group, i.e. R¹ is followed by theintroduction of R² where each of R¹ and R² is alkyl, aralkyl, haloalkylsuch as chloroethyl, or aralkyl (Scheme 32, Reaction 4a) whereby S32.2is converted to S32.1a. Suitable coupling agents are those employed forthe preparation of carboxylate esters, and include a carbodiimide suchas dicyclohexylcarbodiimide, in which case the reaction is preferablyconducted in a basic organic solvent such as pyridine, or(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PYBOP, Sigma), in which case the reaction is performed in a polarsolvent such as dimethylformamide, in the presence of a tertiary organicbase such as diisopropylethylamine, or Aldrithiol-2 (Aldrich) in whichcase the reaction is conducted in a basic solvent such as pyridine, inthe presence of a triaryl phosphine such as triphenylphosphine.Alternatively, the conversion of the phosphonate monoester S32.2 to thediester S32.1 is effected by the use of the Mitsunobu reaction, asdescribed above (Scheme 7). The substrate is reacted with the hydroxycompound R¹OH, in the presence of diethyl azodicarboxylate and atriarylphosphine such as triphenyl phosphine. Alternatively, thephosphonate monoester S32.2 is transformed into the phosphonate diesterS32.1, in which the introduced R¹ group is alkenyl or aralkyl, byreaction of the monoester with the halide R¹Br, in which R¹ is asalkenyl or aralkyl. The alkylation reaction is conducted in a polarorganic solvent such as dimethylformamide or acetonitrile, in thepresence of a base such as cesium carbonate. Alternatively, thephosphonate monoester is transformed into the phosphonate diester in atwo step procedure. In the first step, the phosphonate monoester S32.2is transformed into the chloro analog RP(O)(OR¹)Cl by reaction withthionyl chloride or oxalyl chloride and the like, as described inOrganic Phosphorus Compounds, G. M. Kosolapoff, L. Maeir, eds, Wiley,1976, p. 17, and the thus-obtained product RP(O)(OR¹)Cl is then reactedwith the hydroxy compound R¹OH, in the presence of a base such astriethylamine, to afford the phosphonate diester S32.1.

A phosphonic acid R-link-P(O)(OH)₂ is transformed into a phosphonatemonoester RP(O)(OR¹)(OH) (Scheme 32, Reaction 5) by means of the methodsdescribed above of for the preparation of the phosphonate diesterR-link-P(O)(OR¹)₂ S32.1, except that only one molar proportion of thecomponent R¹OH or R¹Br is employed. Dialkyl phosphonates may be preparedaccording to the methods of: Quast et al (1974) Synthesis 490; Stowellet al (1990) Tetrahedron Lett. 3261; U.S. Pat. No. 5,663,159.

A phosphonic acid R-link-P(O)(OH)₂ S32.3 is transformed into aphosphonate diester R-link-P(O)(OR¹)₂ S32.1 (Scheme 32, Reaction 6) by acoupling reaction with the hydroxy compound R¹OH, in the presence of acoupling agent such as Aldrithiol-2 (Aldrich) and triphenylphosphine.The reaction is conducted in a basic solvent such as pyridine.Alternatively, phosphonic acids S32.3 are transformed into phosphonicesters S32.1 in which R¹ is aryl, by means of a coupling reactionemploying, for example, dicyclohexylcarbodiimide in pyridine at ca 70°C. Alternatively, phosphonic acids S32.3 are transformed into phosphonicesters S32.1 in which R¹ is alkenyl, by means of an alkylation reaction.The phosphonic acid is reacted with the alkenyl bromide R¹Br in a polarorganic solvent such as acetonitrile solution at reflux temperature, thepresence of a base such as cesium carbonate, to afford the phosphonicester S32.1.

Preparation of Phosphonate Carbamates.

Phosphonate esters may contain a carbamate linkage. The preparation ofcarbamates is described in Comprehensive Organic Functional GroupTransformations, A. R. Katritzky, ed., Pergamon, 1995, Vol. 6, p. 416ff,and in Organic Functional Group Preparations, by S. R. Sandler and W.Karo, Academic Press, 1986, p. 260ff. The carbamoyl group may be formedby reaction of a hydroxy group according to the methods known in theart, including the teachings of Ellis, US 2002/0103378 A1 and Hajima, US6018049.

Scheme 33 illustrates various methods by which the carbamate linkage issynthesized. As shown in Scheme 33, in the general reaction generatingcarbamates, an alcohol S33.1, is converted into the activated derivativeS33.2 in which Lv is a leaving group such as halo, imidazolyl,benztriazolyl and the like, as described herein. The activatedderivative S33.2 is then reacted with an amine S33.3, to afford thecarbamate product S33.4. Examples 1-7 in Scheme 33 depict methods bywhich the general reaction is effected. Examples 8-10 illustratealternative methods for the preparation of carbamates.

Scheme 33, Example 1 illustrates the preparation of carbamates employinga chloroformyl derivative of the alcohol S33.5. In this procedure, thealcohol S33.5 is reacted with phosgene, in an inert solvent such astoluene, at about 0° C., as described in Org. Syn. Coll. Vol. 3, 167,1965, or with an equivalent reagent such as trichloromethoxychloroformate, as described in Org. Syn. Coll. Vol. 6, 715, 1988, toafford the chloroformate S33.6. The latter compound is then reacted withthe amine component S33.3, in the presence of an organic or inorganicbase, to afford the carbamate S33.7. For example, the chloroformylcompound S33.6 is reacted with the amine S33.3 in a water-misciblesolvent such as tetrahydrofuran, in the presence of aqueous sodiumhydroxide, as described in Org. Syn. Coll. Vol. 3, 167, 1965, to yieldthe carbamate S33.7. Alternatively, the reaction is performed indichloromethane in the presence of an organic base such asdiisopropylethylamine or dimethylaminopyridine.

Scheme 33, Example 2 depicts the reaction of the chloroformate compoundS33.6 with imidazole to produce the imidazolide S33.8. The imidazolideproduct is then reacted with the amine S33.3 to yield the carbamateS33.7. The preparation of the imidazolide is performed in an aproticsolvent such as dichloromethane at 0′, and the preparation of thecarbamate is conducted in a similar solvent at ambient temperature,optionally in the presence of a base such as dimethylaminopyridine, asdescribed in J. Med. Chem., 1989, 32, 357.

Scheme 33 Example 3, depicts the reaction of the chloroformate S33.6with an activated hydroxyl compound R″OH, to yield the mixed carbonateester S33.10. The reaction is conducted in an inert organic solvent suchas ether or dichloromethane, in the presence of a base such asdicyclohexylamine or triethylamine. The hydroxyl component R″OH isselected from the group of compounds S33.19-S33.24 shown in Scheme 33,and similar compounds. For example, if the component R″OH ishydroxybenztriazole S33.19, N-hydroxysuccinimide S33.20, orpentachlorophenol, S33.21, the mixed carbonate S33.10 is obtained by thereaction of the chloroformate with the hydroxyl compound in an etherealsolvent in the presence of dicyclohexylamine, as described in Can. J.Chem., 1982, 60, 976. A similar reaction in which the component R″OH ispentafluorophenol S33.22 or 2-hydroxypyridine S33.23 is performed in anethereal solvent in the presence of triethylamine, as described in Syn.,1986, 303, and Chem. Ber. 118, 468, 1985.

Scheme 33 Example 4 illustrates the preparation of carbamates in whichan alkyloxycarbonylimidazole S33.8 is employed. In this procedure, analcohol S33.5 is reacted with an equimolar amount of carbonyldiimidazole S33.11 to prepare the intermediate S33.8. The reaction isconducted in an aprotic organic solvent such as dichloromethane ortetrahydrofuran. The acyloxyimidazole

S33.8 is then reacted with an equimolar amount of the amine R′NH₂ toafford the carbamate S33.7. The reaction is performed in an aproticorganic solvent such as dichloromethane, as described in Tet. Lett., 42,2001, 5227, to afford the carbamate S33.7.

Scheme 33, Example 5 illustrates the preparation of carbamates by meansof an intermediate alkoxycarbonylbenztriazole S33.13. In this procedure,an alcohol ROH is reacted at ambient temperature with an equimolaramount of benztriazole carbonyl chloride S33.12, to afford thealkoxycarbonyl product S33.13. The reaction is performed in an organicsolvent such as benzene or toluene, in the presence of a tertiaryorganic amine such as triethylamine, as described in Synthesis., 1977,704. The product is then reacted with the amine R′NH₂ to afford thecarbamate S33.7. The reaction is conducted in toluene or ethanol, atfrom ambient temperature to about 80° C. as described in Synthesis.,1977, 704.

Scheme 33, Example 6 illustrates the preparation of carbamates in whicha carbonate (R″O)₂CO, S33.14, is reacted with an alcohol S33.5 to affordthe intermediate alkyloxycarbonyl intermediate S33.15. The latterreagent is then reacted with the amine R′NH₂ to afford the carbamateS33.7. The procedure in which the reagent S33.15 is derived fromhydroxybenztriazole S33.19 is described in Synthesis, 1993, 908; theprocedure in which the reagent S33.15 is derived fromN-hydroxysuccinimide S33.20 is described in Tet. Lett., 1992, 2781; theprocedure in which the reagent S33.15 is derived from 2-hydroxypyridineS33.23 is described in Tet. Lett., 1991, 4251; the procedure in whichthe reagent S33.15 is derived from 4-nitrophenol S33.24 is described inSynthesis. 1993, 103. The reaction between equimolar amounts of thealcohol ROH and the carbonate S33.14 is conducted in an inert organicsolvent at ambient temperature.

Scheme 33, Example 7 illustrates the preparation of carbamates fromalkoxycarbonyl azides S33.16. In this procedure, an alkyl chloroformateS33.6 is reacted with an azide, for example sodium azide, to afford thealkoxycarbonyl azide S33.16. The latter compound is then reacted with anequimolar amount of the amine R′NH₂ to afford the carbamate S33.7. Thereaction is conducted at ambient temperature in a polar aprotic solventsuch as dimethylsulfoxide, for example as described in Synthesis., 1982,404.

Scheme 33, Example 8 illustrates the preparation of carbamates by meansof the reaction between an alcohol ROH and the chloroformyl derivativeof an amine S33.17. In this procedure, which is described in SyntheticOrganic Chemistry, R. B. Wagner, H. D. Zook, Wiley, 1953, p. 647, thereactants are combined at ambient temperature in an aprotic solvent suchas acetonitrile, in the presence of a base such as triethylamine, toafford the carbamate S33.7.

Scheme 33, Example 9 illustrates the preparation of carbamates by meansof the reaction between an alcohol ROH and an isocyanate S33.18. In thisprocedure, which is described in Synthetic Organic Chemistry, R. B.Wagner, H. D. Zook, Wiley, 1953, p. 645, the reactants are combined atambient temperature in an aprotic solvent such as ether ordichloromethane and the like, to afford the carbamate S33.7.

Scheme 33, Example 10 illustrates the preparation of carbamates by meansof the reaction between an alcohol ROH and an amine R′NH₂. In thisprocedure, which is described in Chem. Lett. 1972, 373, the reactantsare combined at ambient temperature in an aprotic organic solvent suchas tetrahydrofuran, in the presence of a tertiary base such astriethylamine, and selenium. Carbon monoxide is passed through thesolution and the reaction proceeds to afford the carbamate S33.7.

Preparation of Carboalkoxy-Substituted Phosphonate Bisamidates,Monoamidates, Diesters and Monoesters.

A number of methods are available for the conversion of phosphonic acidsinto amidates and esters. In one group of methods, the phosphonic acidis either converted into an isolated activated intermediate such as aphosphoryl chloride, or the phosphonic acid is activated in situ forreaction with an amine or a hydroxy compound.

The conversion of phosphonic acids into phosphoryl chlorides isaccomplished by reaction with thionyl chloride, for example as describedin J. Gen. Chem. USSR, 1983, 53, 480, Zh. Obschei Khim., 1958, 28, 1063,or J. Org. Chem., 1994, 59, 6144, or by reaction with oxalyl chloride,as described in J. Am. Chem. Soc., 1994, 116, 3251, or J. Org. Chem.,1994, 59, 6144, or by reaction with phosphorus pentachloride, asdescribed in J. Org. Chem., 2001, 66, 329, or in J. Med. Chem., 1995,38, 1372. The resultant phosphoryl chlorides are then reacted withamines or hydroxy compounds in the presence of a base to afford theamidate or ester products.

Phosphonic acids are converted into activated imidazolyl derivatives byreaction with carbonyl diimidazole, as described in J. Chem. Soc., Chem.Comm. (1991) 312, or Nucleosides & Nucleotides (2000) 19:1885. Activatedsulfonyloxy derivatives are obtained by the reaction of phosphonic acidswith trichloromethylsulfonyl chloride or withtriisopropylbenzenesulfonyl chloride, as described in Tet. Lett. (1996)7857, or Bioorg. Med. Chem. Lett. (1998) 8:663. The activatedsulfonyloxy derivatives are then reacted with amines or hydroxycompounds to afford amidates or esters.

Alternatively, the phosphonic acid and the amine or hydroxy reactant arecombined in the presence of a diimide coupling agent. The preparation ofphosphonic amidates and esters by means of coupling reactions in thepresence of dicyclohexyl carbodiimide is described, for example, in J.Chem. Soc., Chem. Comm. (1991) 312 or Coll. Czech. Chem. Comm. (1987)52:2792. The use of ethyl dimethylaminopropyl carbodiimide foractivation and coupling of phosphonic acids is described in Tet. Lett.,(2001) 42:8841, or Nucleosides & Nucleotides (2000) 19:1885.

A number of additional coupling reagents have been described for thepreparation of amidates and esters from phosphonic acids. The agentsinclude Aldrithiol-2, and PYBOP and BOP, as described in J. Org. Chem.,1995, 60, 5214, and J. Med. Chem. (1997) 40:3842,mesitylene-2-sulfonyl-3-nitro-1,2,4-triazole (MSNT), as described in J.Med. Chem. (1996) 39:4958, diphenylphosphoryl azide, as described in J.Org. Chem. (1984) 49:1158,1-(2,4,6-triisopropylbenzenesulfonyl-3-nitro-1,2,4-triazole (TPSNT) asdescribed in Bioorg. Med. Chem. Lett. (1998) 8:1013,bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), asdescribed in Tet. Lett., (1996) 37:3997,2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, as described inNucleosides Nucleotides 1995, 14, 871, and diphenyl chlorophosphate, asdescribed in J. Med. Chem., 1988, 31, 1305.

Phosphonic acids are converted into amidates and esters by means of theMitsunobu reaction, in which the phosphonic acid and the amine orhydroxy reactant are combined in the presence of a triaryl phosphine anda dialkyl azodicarboxylate. The procedure is described in Org. Lett.,2001, 3, 643, or J. Med. Chem., 1997, 40, 3842.

Phosphonic esters are also obtained by the reaction between phosphonicacids and halo compounds, in the presence of a suitable base. The methodis described, for example, in Anal. Chem., 1987, 59, 1056, or J. Chem.Soc. Perkin Trans., I, 1993, 19, 2303, or J. Med. Chem., 1995, 38, 1372,or Tet. Lett., 2002, 43, 1161.

Schemes 34-37 illustrate the conversion of phosphonate esters andphosphonic acids into carboalkoxy-substituted phosphonbisamidates(Scheme 34), phosphonamidates (Scheme 35), phosphonate monoesters(Scheme 36) and phosphonate diesters, (Scheme 37). Scheme 38 illustratessynthesis of gem-dialkyl amino phosphonate reagents.

Scheme 34 illustrates various methods for the conversion of phosphonatediesters S34.1 into phosphonbisamidates S34.5. The diester S34.1,prepared as described previously, is hydrolyzed, either to the monoesterS34.2 or to the phosphonic acid S34.6. The methods employed for thesetransformations are described above. The monoester S34.2 is convertedinto the monoamidate S34.3 by reaction with an aminoester S34.9, inwhich the group R² is H or alkyl; the group R^(4b) is a divalentalkylene moiety such as, for example, CHCH₃, CHCH₂CH₃, CH(CH(CH₃)₂),CH(CH₂Ph), and the like, or a side chain group present in natural ormodified aminoacids; and the group R^(5b) is C₁-C₁₂ alkyl, such asmethyl, ethyl, propyl, isopropyl, or isobutyl; C₆-C₂₀ aryl, such asphenyl or substituted phenyl; or C₆-C₂₀ arylalkyl, such as benzyl orbenzyhydryl. The reactants are combined in the presence of a couplingagent such as a carbodiimide, for example dicyclohexyl carbodiimide, asdescribed in J. Am. Chem. Soc., (1957) 79:3575, optionally in thepresence of an activating agent such as hydroxybenztriazole, to yieldthe amidate product S34.3. The amidate-forming reaction is also effectedin the presence of coupling agents such as BOP, as described in J. Org.Chem. (1995) 60:5214, Aldrithiol, PYBOP and similar coupling agents usedfor the preparation of amides and esters. Alternatively, the reactantsS34.2 and S34.9 are transformed into the monoamidate S34.3 by means of aMitsunobu reaction. The preparation of amidates by means of theMitsunobu reaction is described in J. Med. Chem. (1995) 38:2742.Equimolar amounts of the reactants are combined in an inert solvent suchas tetrahydrofuran in the presence of a triaryl phosphine and a dialkylazodicarboxylate. The thus-obtained monoamidate ester S34.3 is thentransformed into amidate phosphonic acid S34.4. The conditions used forthe hydrolysis reaction depend on the nature of the R¹ group, asdescribed previously. The phosphonic acid amidate S34.4 is then reactedwith an aminoester S34.9, as described above, to yield the bisamidateproduct S34.5, in which the amino substituents are the same ordifferent. Alternatively, the phosphonic acid S34.6 may be treated withtwo different amino ester reagents simulataneously, i.e. S34.9 where R²,R^(4b) or R^(5b) are different. The resulting mixture of bisamidateproducts S34.5 may then be separable, e.g. by chromatography.

An example of this procedure is shown in Scheme 34, Example 1. In thisprocedure, a dibenzyl phosphonate S34.14 is reacted withdiazabicyclooctane (DABCO) in toluene at reflux, as described in J. Org.Chem., 1995, 60, 2946, to afford the monobenzyl phosphonate S34.15. Theproduct is then reacted with equimolar amounts of ethyl alaninate S34.16and dicyclohexyl carbodiimide in pyridine, to yield the amidate productS34.17. The benzyl group is then removed, for example by hydrogenolysisover a palladium catalyst, to give the monoacid product S34.18 which maybe unstable according to J. Med. Chem. (1997) 40(23):3842. This compoundS34.18 is then reacted in a Mitsunobu reaction with ethyl leucinateS34.19, triphenyl phosphine and diethylazodicarboxylate, as described inJ. Med. Chem., 1995, 38, 2742, to produce the bisamidate product S34.20.

Using the above procedures, but employing in place of ethyl leucinate

S34.19 or ethyl alaninate S34.16, different aminoesters S34.9, thecorresponding products S34.5 are obtained.

Alternatively, the phosphonic acid S34.6 is converted into thebisamidate S34.5 by use of the coupling reactions described above. Thereaction is performed in one step, in which case the nitrogen-relatedsubstituents present in the product S34.5 are the same, or in two steps,in which case the nitrogen-related substituents can be different.

An example of the method is shown in Scheme 34, Example 2. In thisprocedure, a phosphonic acid S34.6 is reacted in pyridine solution withexcess ethyl phenylalaninate S34.21 and dicyclohexylcarbodiimide, forexample as described in J. Chem. Soc., Chem. Comm., 1991, 1063, to givethe bisamidate product S34.22.

Using the above procedures, but employing, in place of ethylphenylalaninate, different aminoesters S34.9, the corresponding productsS34.5 are obtained.

As a further alternative, the phosphonic acid S34.6 is converted intothe mono or bis-activated derivative S34.7, in which Lv is a leavinggroup such as chloro, imidazolyl, triisopropylbenzenesulfonyloxy etc.The conversion of phosphonic acids into chlorides S34.7 (Lv=Cl) iseffected by reaction with thionyl chloride or oxalyl chloride and thelike, as described in Organic Phosphorus Compounds, G. M. Kosolapoff, L.Macir, eds, Wiley, 1976, p. 17. The conversion of phosphonic acids intomonoimidazolides S34.7 (Lv=imidazolyl) is described in J. Med. Chem.,2002, 45, 1284 and in J. Chem. Soc. Chem. Comm., 1991, 312.Alternatively, the phosphonic acid is activated by reaction withtriisopropylbenzenesulfonyl chloride, as described in Nucleosides andNucleotides, 2000, 10, 1885. The activated product is then reacted withthe aminoester S34.9, in the presence of a base, to give the bisamidateS34.5. The reaction is performed in one step, in which case the nitrogensubstituents present in the product S34.5 are the same, or in two steps,via the intermediate S34.11, in which case the nitrogen substituents canbe different.

Examples of these methods are shown in Scheme 34, Examples 3 and 5. Inthe procedure illustrated in Scheme 34, Example 3, a phosphonic acidS34.6 is reacted with ten molar equivalents of thionyl chloride, asdescribed in Zh. Obschei Khim., 1958, 28, 1063, to give the dichlorocompound S34.23. The product is then reacted at reflux temperature in apolar aprotic solvent such as acetonitrile, and in the presence of abase such as triethylamine, with butyl serinate S34.24 to afford thebisamidate product S34.25.

Using the above procedures, but employing, in place of butyl serinateS34.24, different aminoesters S34.9, the corresponding products S34.5are obtained.

In the procedure illustrated in Scheme 34, Example 5, the phosphonicacid S34.6 is reacted, as described in J. Chem. Soc. Chem. Comm., 1991,312, with carbonyl diimidazole to give the imidazolide S34.S32. Theproduct is then reacted in acetonitrile solution at ambient temperature,with one molar equivalent of ethyl alaninate S34.33 to yield themonodisplacement product S34.S34. The latter compound is then reactedwith carbonyl diimidazole to produce the activated intermediate S34.35,and the product is then reacted, under the same conditions, with ethylN-methylalaninate S34.33a to give the bisamidate product S34.36.

Using the above procedures, but employing, in place of ethyl alaninateS34.33 or ethyl N-methylalaninate S34.33a, different aminoesters S34.9,the corresponding products S34.5 are obtained.

The intermediate monoamidate S34.3 is also prepared from the monoesterS34.2 by first converting the monoester into the activated derivativeS34.8 in which Lv is a leaving group such as halo, imidazolyl etc, usingthe procedures described above. The product S34.8 is then reacted withan aminoester S34.9 in the presence of a base such as pyridine, to givean intermediate monoamidate product S34.3. The latter compound is thenconverted, by removal of the R¹ group and coupling of the product withthe aminoester S34.9, as described above, into the bisamidate S34.5.

An example of this procedure, in which the phosphonic acid is activatedby conversion to the chloro derivative S34.26, is shown in Scheme 34,Example 4. In this procedure, the phosphonic monobenzyl ester S34.15 isreacted, in dichloromethane, with thionyl chloride, as described in Tet.Letters., 1994, 35, 4097, to afford the phosphoryl chloride S34.26. Theproduct is then reacted in acetonitrile solution at ambient temperaturewith one molar equivalent of ethyl 3-amino-2-methylpropionate S34.27 toyield the monoamidate product S34.28. The latter compound ishydrogenated in ethylacetate over a 5% palladium on carbon catalyst toproduce the monoacid product S34.29. The product is subjected to aMitsunobu coupling procedure, with equimolar amounts of butyl alaninateS34.30, triphenyl phosphine, diethylazodicarboxylate and triethylaminein tetrahydrofuran, to give the bisamidate product S34.31.

Using the above procedures, but employing, in place of ethyl3-amino-2-methylpropionate S34.27 or butyl alaninate S34.30, differentaminoesters S34.9, the corresponding products S34.5 are obtained.

The activated phosphonic acid derivative S34.7 is also converted intothe bisamidate S34.5 via the diamino compound S34.10. The conversion ofactivated phosphonic acid derivatives such as phosphoryl chlorides intothe corresponding amino analogs S34.10, by reaction with ammonia, isdescribed in Organic Phosphorus Compounds, G. M. Kosolapoff, L. Maeir,eds, Wiley, 1976. The bisamino compound S34.10 is then reacted atelevated temperature with a haloester S34.12 (Hal=halogen, i.e. F, Cl,Br, I), in a polar organic solvent such as dimethylformamide, in thepresence of a base such as 4,4-dimethylaminopyridine (DMAP) or potassiumcarbonate, to yield the bisamidate S34.5. Alternatively, S34.6 may betreated with two different amino ester reagents simulataneously, i.e.S34.12 where R^(4b) or R^(5b) are different. The resulting mixture ofbisamidate products S34.5 may then be separable, e.g. by chromatography.

An example of this procedure is shown in Scheme 34, Example 6. In thismethod, a dichlorophosphonate S34.23 is reacted with ammonia to affordthe diamide S34.37. The reaction is performed in aqueous, aqueousalcoholic or alcoholic solution, at reflux temperature. The resultingdiamino compound is then reacted with two molar equivalents of ethyl2-bromo-3-methylbutyrate S34.38, in a polar organic solvent such asN-methylpyrrolidinone at ca. 150° C., in the presence of a base such aspotassium carbonate, and optionally in the presence of a catalyticamount of potassium iodide, to afford the bisamidate product S34.39.

Using the above procedures, but employing, in place of ethyl2-bromo-3-methylbutyrate S34.38, different haloesters S34.12 thecorresponding products S34.5 are obtained.

The procedures shown in Scheme 34 are also applicable to the preparationof bisamidates in which the aminoester moiety incorporates differentfunctional groups. Scheme 34, Example 7 illustrates the preparation ofbisamidates derived from tyrosine. In this procedure, themonoimidazolide S34.32 is reacted with propyl tyrosinate S34.40, asdescribed in Example 5, to yield the monoamidate S34.41. The product isreacted with carbonyl diimidazole to give the imidazolide S34.42, andthis material is reacted with a further molar equivalent of propyltyrosinate to produce the bisamidate product S34.43.

Using the above procedures, but employing, in place of propyl tyrosinateS34.40, different aminoesters S34.9, the corresponding products S34.5are obtained. The aminoesters employed in the two stages of the aboveprocedure can be the same or different, so that bisamidates with thesame or different amino substituents are prepared.

Scheme 35 illustrates methods for the preparation of phosphonatemonoamidates.

In one procedure, a phosphonate monoester S34.1 is converted, asdescribed in Scheme 34, into the activated derivative S34.8. Thiscompound is then reacted, as described above, with an aminoester S34.9,in the presence of a base, to afford the monoamidate product S35.1.

The procedure is illustrated in Scheme 35, Example 1. In this method, amonophenyl phosphonate S35.7 is reacted with, for example, thionylchloride, as described in J. Gen. Chem. USSR., 1983, 32, 367, to givethe chloro product S35.8. The product is then reacted, as described inScheme 34, with ethyl alaninate S3, to yield the amidate S35.10.

Using the above procedures, but employing, in place of ethyl alaninateS35.9, different aminoesters S34.9, the corresponding products S35.1 areobtained.

Alternatively, the phosphonate monoester S34.1 is coupled, as describedin Scheme 34, with an aminoester S34.9 to produce the amidate S335.1. Ifnecessary, the R¹ substituent is then altered, by initial cleavage toafford the phosphonic acid S35.2. The procedures for this transformationdepend on the nature of the R¹ group, and are described above. Thephosphonic acid is then transformed into the ester amidate productS35.3, by reaction with the hydroxy compound R³OH, in which the group R³is aryl, heterocycle, alkyl, cycloalkyl, haloalkyl etc, using the samecoupling procedures (carbodiimide, Aldrithiol-2, PYBOP, Mitsunobureaction etc) described in Scheme 34 for the coupling of amines andphosphonic acids.

Examples of this method are shown in Scheme 35, Examples and 2 and 3. Inthe sequence shown in Example 2, a monobenzyl phosphonate S35.11 istransformed by reaction with ethyl alaninate, using one of the methodsdescribed above, into the monoamidate S35.12. The benzyl group is thenremoved by catalytic hydrogenation in ethylacetate solution over a 5%palladium on carbon catalyst, to afford the phosphonic acid amidateS35.13. The product is then reacted in dichloromethane solution atambient temperature with equimolar amounts of1-(dimethylaminopropyl)-3-ethylcarbodiimide and trifluoroethanol S35.14,for example as described in Tet. Lett., 2001, 42, 8841, to yield theamidate ester S35.15.

In the sequence shown in Scheme 35, Example 3, the monoamidate S35.13 iscoupled, in tetrahydrofuran solution at ambient temperature, withequimolar amounts of dicyclohexyl carbodiimide and4-hydroxy-N-methylpiperidine S35.16, to produce the amidate esterproduct S35.17.

Using the above procedures, but employing, in place of the ethylalaninate product S35.12 different monoacids S35.2, and in place oftrifluoroethanol S35.14 or 4-hydroxy-N-methylpiperidine S35.16,different hydroxy compounds R³OH, the corresponding products S35.3 areobtained.

Alternatively, the activated phosphonate ester S34.8 is reacted withammonia to yield the amidate S35.4. The product is then reacted, asdescribed in Scheme 34, with a haloester S35.5, in the presence of abase, to produce the amidate product S35.6. If appropriate, the natureof the R¹ group is changed, using the procedures described above, togive the product S35.3. The method is illustrated in Scheme 35, Example4. In this sequence, the monophenyl phosphoryl chloride S35.18 isreacted, as described in Scheme 34, with ammonia, to yield the aminoproduct S35.19. This material is then reacted in N-methylpyrrolidinonesolution at 170° with butyl 2-bromo-3-phenylpropionate S35.20 andpotassium carbonate, to afford the amidate product S35.21.

Using these procedures, but employing, in place of butyl2-bromo-3-phenylpropionate S35.20, different haloesters S35.5, thecorresponding products S35.6 are obtained.

The monoamidate products S35.3 are also prepared from the doublyactivated phosphonate derivatives S34.7. In this procedure, examples ofwhich are described in Synlett., 1998, 1, 73, the intermediate S34.7 isreacted with a limited amount of the aminoester S34.9 to give themono-displacement product S34.11. The latter compound is then reactedwith the hydroxy compound R³OH in a polar organic solvent such asdimethylformamide, in the presence of a base such asdiisopropylethylamine, to yield the monoamidate ester S35.3.

The method is illustrated in Scheme 35, Example 5. In this method, thephosphoryl dichloride S35.22 is reacted in dichloromethane solution withone molar equivalent of ethyl N-methyl tyrosinate S35.23 anddimethylaminopyridine, to generate the monoamidate S35.24. The productis then reacted with phenol S35.25 in dimethylformamide containingpotassium carbonate, to yield the ester amidate product S35.26.

Using these procedures, but employing, in place of ethyl N-methyltyrosinate S35.23 or phenol S35.25, the aminoesters 34.9 and/or thehydroxy compounds R³OH, the corresponding products S35.3 are obtained.

Scheme 36 illustrates methods for the preparation ofcarboalkoxy-substituted phosphonate diesters in which one of the estergroups incorporates a carboalkoxy substituent.

In one procedure, a phosphonate monoester S34.1, prepared as describedabove, is coupled, using one of the methods described above, with ahydroxyester S36.1, in which the groups R^(4b) and R^(5b) are asdescribed in Scheme 34. For example, equimolar amounts of the reactantsare coupled in the presence of a carbodiimide such as dicyclohexylcarbodiimide, as described in Aust. J. Chem., 1963, 609, optionally inthe presence of dimethylaminopyridine, as described in Tet., 1999, 55,12997. The reaction is conducted in an inert solvent at ambienttemperature.

The procedure is illustrated in Scheme 36, Example 1. In this method, amonophenyl phosphonate S36.9 is coupled, in dichloromethane solution inthe presence of dicyclohexyl carbodiimide, with ethyl3-hydroxy-2-methylpropionate S36.10 to yield the phosphonate mixeddiester S36.11.

Using this procedure, but employing, in place of ethyl3-hydroxy-2-methylpropionate S36.10, different hydroxyesters S33.1, thecorresponding products S33.2 are obtained.

The conversion of a phosphonate monoester S34.1 into a mixed diesterS36.2 is also accomplished by means of a Mitsunobu coupling reactionwith the hydroxyester S36.1, as described in Org. Lett., 2001, 643. Inthis method, the reactants 34.1 and S36.1 are combined in a polarsolvent such as tetrahydrofuran, in the presence of a triarylphosphineand a dialkyl azodicarboxylate, to give the mixed diester S36.2. The R¹substituent is varied by cleavage, using the methods describedpreviously, to afford the monoacid product S36.3. The product is thencoupled, for example using methods described above, with the hydroxycompound R³OH, to give the diester product S36.4.

The procedure is illustrated in Scheme 36, Example 2. In this method, amonoallyl phosphonate S36.12 is coupled in tetrahydrofuran solution, inthe presence of triphenylphosphine and diethylazodicarboxylate, withethyl lactate S36.13 to give the mixed diester S36.14. The product isreacted with tris(triphenylphosphine) rhodium chloride (Wilkinsoncatalyst) in acetonitrile, as described previously, to remove the allylgroup and produce the monoacid product S36.15. The latter compound isthen coupled, in pyridine solution at ambient temperature, in thepresence of dicyclohexyl carbodiimide, with one molar equivalent of3-hydroxypyridine S36.16 to yield the mixed diester S36.17.

Using the above procedures, but employing, in place of the ethyl lactateS36.13 or 3-hydroxypyridine, a different hydroxyester S36.1 and/or adifferent hydroxy compound R³OH, the corresponding products S36.4 areobtained.

The mixed diesters S36.2 are also obtained from the monoesters S34.1 viathe intermediacy of the activated monoesters S36.5. In this procedure,the monoester S34.1 is converted into the activated compound S36.5 byreaction with, for example, phosphorus pentachloride, as described in J.Org. Chem., 2001, 66, 329, or with thionyl chloride or oxalyl chloride(Lv=Cl), or with triisopropylbenzenesulfonyl chloride in pyridine, asdescribed in Nucleosides and Nucleotides, 2000, 19, 1885, or withcarbonyl diimidazole, as described in J. Med. Chem., 2002, 45, 1284. Theresultant activated monoester is then reacted with the hydroxyesterS36.1, as described above, to yield the mixed diester S36.2.

The procedure is illustrated in Scheme 36, Example 3. In this sequence,a monophenyl phosphonate S36.9 is reacted, in acetonitrile solution at70° C., with ten equivalents of thionyl chloride, so as to produce thephosphoryl chloride S36.19. The product is then reacted with ethyl4-carbamoyl-2-hydroxybutyrate S36.20 in dichloromethane containingtriethylamine, to give the mixed diester S36.21.

Using the above procedures, but employing, in place of ethyl4-carbamoyl-2-hydroxybutyrate S36.20, different hydroxyesters S36.1, thecorresponding products S36.2 are obtained.

The mixed phosphonate diesters are also obtained by an alternative routefor incorporation of the R³⁰ group into intermediates S36.3 in which thehydroxyester moiety is already incorporated. In this procedure, themonoacid intermediate S36.3 is converted into the activated derivativeS36.6 in which Lv is a leaving group such as chloro, imidazole, and thelike, as previously described. The activated intermediate is thenreacted with the hydroxy compound R³OH, in the presence of a base, toyield the mixed diester product S36.4.

The method is illustrated in Scheme 36, Example 4. In this sequence, thephosphonate monoacid S36.22 is reacted with trichloromethanesulfonylchloride in tetrahydrofuran containing collidine, as described in J.Med. Chem., 1995, 38, 4648, to produce the trichloromethanesulfonyloxyproduct S36.23. This compound is reacted with 3-(morpholinomethyl)phenolS36.24 in dichloromethane containing triethylamine, to yield the mixeddiester product S36.25.

Using the above procedures, but employing, in place of with3-(morpholinomethyl)phenol S36.24, different alcohols R³OH, thecorresponding products S36.4 are obtained.

The phosphonate esters S36.4 are also obtained by means of alkylationreactions performed on the monoesters S34.1. The reaction between themonoacid S34.1 and the haloester S36.7 is performed in a polar solventin the presence of a base such as diisopropylethylamine, as described inAnal. Chem., 1987, 59, 1056, or triethylamine, as described in J. Med.Chem., 1995, 38, 1372, or in a non-polar solvent such as benzene, in thepresence of 18-crown-6, as described in Syn. Comm., 1995, 25, 3565.

The method is illustrated in Scheme 36, Example 5. In this procedure,the monoacid S36.26 is reacted with ethyl 2-bromo-3-phenylpropionateS36.27 and diisopropylethylamine in dimethylformamide at 80° C. toafford the mixed diester product S36.28.

Using the above procedure, but employing, in place of ethyl2-bromo-3-phenylpropionate S36.27, different haloesters S36.7, thecorresponding products S36.4 are obtained.

Scheme 37 illustrates methods for the preparation of phosphonatediesters in which both the ester substituents incorporate carboalkoxygroups.

The compounds are prepared directly or indirectly from the phosphonicacids S34.6. In one alternative, the phosphonic acid is coupled with thehydroxyester S37.2, using the conditions described previously in Schemes34-36, such as coupling reactions using dicyclohexyl carbodiimide orsimilar reagents, or under the conditions of the Mitsunobu reaction, toafford the diester product S37.3 in which the ester substituents areidentical.

This method is illustrated in Scheme 37, Example 1. In this procedure,the phosphonic acid S34.6 is reacted with three molar equivalents ofbutyl lactate S37.5 in the presence of Aldrithiol-2 and triphenylphosphine in pyridine at ca. 70° C., to afford the diester S37.6.

Using the above procedure, but employing, in place of butyl lactateS37.5, different hydroxyesters S37.2, the corresponding products S37.3are obtained.

Alternatively, the diesters S37.3 are obtained by alkylation of thephosphonic acid S34.6 with a haloester S37.1. The alkylation reaction isperformed as described in Scheme 36 for the preparation of the estersS36.4.

This method is illustrated in Scheme 37, Example 2. In this procedure,the phosphonic acid S34.6 is reacted with excess ethyl3-bromo-2-methylpropionate S37.7 and diisopropylethylamine indimethylformamide at ca. 80° C., as described in Anal. Chem., 1987, 59,1056, to produce the diester S37.8.

Using the above procedure, but employing, in place of ethyl3-bromo-2-methylpropionate S37.7, different haloesters S37.1, thecorresponding products S37.3 are obtained.

The diesters S37.3 are also obtained by displacement reactions ofactivated derivatives S34.7 of the phosphonic acid with thehydroxyesters S37.2. The displacement reaction is performed in a polarsolvent in the presence of a suitable base, as described in Scheme 36.The displacement reaction is performed in the presence of an excess ofthe hydroxyester, to afford the diester product S37.3 in which the estersubstituents are identical, or sequentially with limited amounts ofdifferent hydroxyesters, to prepare diesters S37.3 in which the estersubstituents are different.

The methods are illustrated in Scheme 37, Examples 3 and 4. As shown inExample 3, the phosphoryl dichloride S35.22 is reacted with three molarequivalents of ethyl 3-hydroxy-2-(hydroxymethyl)propionate S37.9 intetrahydrofuran containing potassium carbonate, to obtain the diesterproduct S37.10.

Using the above procedure, but employing, in place of ethyl3-hydroxy-2-(hydroxymethyl)propionate S37.9, different hydroxyestersS37.2, the corresponding products S37.3 are obtained.

Scheme 37, Example 4 depicts the displacement reaction between equimolaramounts of the phosphoryl dichloride S35.22 and ethyl2-methyl-3-hydroxypropionate S37.11, to yield the monoester productS37.12. The reaction is conducted in acetonitrile at 70° in the presenceof diisopropylethylamine. The product S37.12 is then reacted, under thesame conditions, with one molar equivalent of ethyl lactate S37.13, togive the diester product S37.14.

Using the above procedures, but employing, in place of ethyl2-methyl-3-hydroxypropionate S37.11 and ethyl lactate S37.13, sequentialreactions with different hydroxyesters S37.2, the corresponding productsS37.3 are obtained.

2,2-Dimethyl-2-aminoethylphosphonic acid intermediates can be preparedby the route in Scheme 5. Condensation of 2-methyl-2-propanesulfinamidewith acetone give sulfinyl imine S38.11 (J. Org. Chem. 1999, 64, 12).Addition of dimethyl methylphosphonate lithium to S38.11 afford S38.12.Acidic methanolysis of S38.12 provide amine S38.13. Protection of aminewith Cbz group and removal of methyl groups yield phosphonic acidS38.14, which can be converted to desired S38.15 (Scheme 38a) usingmethods reported earlier on. An alternative synthesis of compound S38.14is also shown in Scheme 38b. Commercially available2-amino-2-methyl-1-propanol is converted to aziridines S38.16 accordingto literature methods (J. Org. Chem. 1992, 57, 5813; Syn. Lett. 1997, 8,893). Aziridine opening with phosphite give S38.17 (Tetrahedron Lett.1980, 21, 1623). Reprotection) of S38.17 affords S38.14.

The invention will now be illustrated by the following non-limitingExamples.

Example 1 Synthesis of Representative Compounds of Formula 1

Representative compounds of the invention can be prepared as illustratedabove. Dehydroepiandrosterone (purchased from a supplier such asAldrich), 16-α-bromodehydroepiandrosterone, or16-β-bromodehydroepiandrosterone (purchased from a supplier such asSteraloids) can be treated with bases such as, but not limited to,Cs₂CO₃ or NaH, in an appropriate solvents such as, but not limited to,THF or DMF, in the presence of an alkylating agent of general structure1.3. Note that X is a leaving group, preferably in this casetrifluoromethanesulfonate, but other leaving groups may be used andinclude bromide, iodide, chloride, p-toluenesulfonate, methanesulfonate,among others. The phosphonate esters of the resulting alkylated product1.4 can then be converted into the intended final phosphonatefunctionality.

For instance, dehydroepiandrosterone can be treated in anhydrous THF at0° C. with NaH. When bubbling ceases, diethyl phosphonomethyltriflate(prepared according to Tetrahedron Lett. 1986, 27, 1477) is addedyielding intermediate 1.5. The phosphonate esters of 1.5 are thenconverted into the final desired functionality.

Example 2 Synthesis of Representative Compounds of Formula 2

Representative compounds of the invention can be prepared as illustratedabove. Intermediates 2.2 are prepared according to the methods describedin U.S. Pat. No. 6,194,398 and any literature cited therein. Thephosphonate ester of 2.2 may be converted to the final desiredphosphonic acid functionality. Alternatively, phosphonic acids 2.3 maybe formed by cleavage of esters 2.2 by treatment with a reagent such as,but not limited to, TMS-bromide in a solvent such as MeCN. Phosphonicacid 2.3 may then be converted to the final desired phosphonic acidfunctionality.

For instance, LY-582563, prepared as described in U.S. Pat. No.6,194,398 is treated with TMS-Br and 2,6-lutidine in MeCN to providephosphonic acid 2.4. Either LY-582563 or 2.4 may then be converted tothe final desired phosphonate derivative.

Example 3 Synthesis of Representative Compounds of Formulae 3 and 4

Representative compounds of the invention can be prepared as illustratedabove. L-Fd4C and L-FddC are prepared according to methods in U.S. Pat.No. 5,561,120, U.S. Pat. No. 5,627,160, and U.S. Pat. No. 5,631,239 andany literature references cited therein. Either can be treated with abase such as, but not limited to, NaH or Cs₂CO₃, in a solvent such as,but not limited to, THF or DMF, and an alkylating agent of structure3.5. In compounds 3.5, X is a leaving group such as, but not limited to,bromide, chloride, iodide, p-toluenesulfonate,trifluoromethanesulfonate, or methanesulfonate. It should be noted thatcytosine-containing compounds sometimes require protection of the aminogroup at the 4-position of the base. If necessary, a protecting groupmay be introduced onto this position before these alkylation reactionsare carried out. Introduction of such protecting groups (and theirsubsequent removal at the end of a synthetic scheme) are processes wellknown to those skilled in the art of nucleoside and nucleotidesynthesis.

For instance, L-FddC is treated with NaH in DMF at 0° C. When bubblinghas ceased, diethyl phosphonomethyltriflate (prepared according toTetrahedron Lett. 1986, 27, 1477) is added. The resulting product 3.8 isisolated by standard chromatographic means. It may be necessary toprotect the amino group at the 4-position of the base before thisalkylation is carried out. See the note above regarding such protectinggroups.

Example 4 Synthesis of Representative Compounds of Formulae 5 and 6

In Example 4, glycal 4.9 (obtained as described in J. Am. Chem. Soc.1972, 94, 3213) is reacted with phenylselenyl chloride followed bytreatment with the respective phosphonate alcohols 4.10 in the presenceof silver perchlorate (J. Org. Chem. 1991, 56, 2642-2647). Oxidation ofthe resulting chloride using hydrogen peroxide followed by aminolysis ofuracil using triazole, 2-chlorophenyldichlorophosphate, pyridine andammonia (Bioorg. Med. Chem. Lett. 1997, 7, 2567) provides the L-Fd4Cphosphonate derivative 4.12. Hydrogenation over 10% Pd/C provides theL-FddC derivative 4.13.

For instance, glycal 4.9 is reacted with phenylselenyl chloride and thentreated with AgC₄ and diethyl phosphonomethanol (available from Aldrich)providing compound 4.14. Treatment of 4.14 with H₂O₂ and NaHCO₃ in1,4-dioxane followed by triazole, 2-chlorophenyldichlorophospate, inpyridine with ammonia yields the fluorocytosine derivative 4.15.Hydrogenation at 1 atm, over 10% Pd/C yields derivative 4.16.

Example 5 Synthesis of Representative Compounds of Formula 9

-   -   Bases such as but not limited to, thymine, adenine, uracil,        5-halouracils, 5-alkyluracils, guanine, cytosine, 5-halo and        alkyl cytosines, 2,6-diaminopurine. Bases requiring protecting        groups are to be suitably protected using protecting groups and        conditions well known to those skilled in the art.

Representative compounds of the invention can be prepared as illustratedabove. Compounds 5.4, prepared as described in WO 00/09531, U.S. Pat.No. 6,395,716, and U.S. Pat. No. 6,444,652, can be converted to glycal5.11 according to the process reported in J. Am. Chem. Soc. 1972, 94,3213. Glycal 5.11 is then treated with IBr in the presence of alcohol5.12 to provide intermediate 5.13 (see J. Org. Chem. 1991, 56, 2642).The iodide of intermediate 5.13 can be treated with AgOAc to provideacetate 5.14, which can be deacetylated in the presence of catalyticsodium methoxide in methanol. Treatment of this product with DEAD andPPh₃ in the presence of acetic acid, followed by another deprotectionwith catalytic sodium methoxide in methanol will provide intermediate5.15, which is representative of Formula 9. The phosphonates ofintermediates 5.15 can be converted into other embodiments of theinvention according to procedures know to those of skill in the art.

For instance, compound 5.8 is converted into glycal 5.16 according tothe procedures reported in J. Am. Chem. Soc. 1972, 94, 3213. Glycal 5.16is then treated with IBr in the presence of diethyl phosphonomethanol toprovide intermediate 5.17 (see J. Org. Chem. 1991, 56, 2642).Intermediate 5.17 is then treated with AgOAc followed by deprotectionwith catalytic NaOMe in MeOH to provide 5.18. This compound is thenconverted into epimer 5.19 by a Mitsunobu reaction with DEAD/PPh₃ andHOAc in THF, followed by a second catalytic NaOMe/MeOH deprotection. Atany point in the synthetic sequence where it is appropriate, thephosphonate group may be converted into a phosphonate with the desiredsubstitution.

Example 6 Synthesis of Representative Compounds of Formulae 10 and 11

Representative compounds of the invention can be prepared as illustratedabove. The preparation of compounds of structural type 6.6 are describedin U.S. Pat. No. 5,565,438, U.S. Pat. No. 5,567,688, and U.S. Pat. No.5,587,362, and the references cited therein. The compounds are thentreated with a limiting amount of NaH in an appropriate solvent such as,but not limited to THF or DMF, and are then treated with an alkylatingagent of type 6.7 (X=leaving group such as, but not limited to bromide,chloride, iodide, methanesulfonate, trifluoromethanesulfonate, andp-toluenesulfonate). Intermediates 6.8 and 6.9 result as a mixture andcan be separated by chromatographic means that are well known to thoseskilled in the art. It should be noted that if a base requires aprotecting group during this alkylation reaction, suitable protectinggroups either will have already been installed throughout the syntheticschemes that provided starting materials 6.6 described in the citedpatents, or can be installed prior to the alkylation reaction accordingto methods well known to chemists skilled in the art. If a protectinggroup had been added, it may be cleaved at this time according to themethods described in the patents cited above or according to anyappropriate method known to those skilled in the art. At this point, thephosphonate esters may be converted to the desired final phosphonatefunctionality.

Clevudine, prepared as described in the patents cited above, is treatedin anhydrous THF with NaH at 0° C. When bubbling ceases, diethylphosphonomethyltriflate (prepared as in Tetrahedron Lett. 1986, 27,1477) is added. The resulting alkylation products 6.10 and 6.11 areisolated after work-up either using silica gel or reversed-phasechromatography. The phosphonates may then be converted to the finaldesired products.

Example 7 Synthesis of Representative Compounds of Formula 12

Representative compounds of the invention can be prepared as illustratedabove. L-Deoxynucleoside 7.12 is synthesized according to literatureprocedure (see the methods reported by Holy, Collect. Czech. Chem.Commun. 1972, 37, 4072). L-Deoxynucleoside 7.12 is then converted into7.13 through the procedures reported in J. Am. Chem. Soc. 1972, 94, 3213and J. Org. Chem. 1991, 56, 2642. Dimethyl phosphonomethanol may bereplaced with any alcohol linked to a phosphonate. The double bond ofcompound 7.13 is then treated with OsO₄ and N-methylmorpholine N-oxideto provide the dihydroxylated derivatives 7.14. Triflation of 7.14results in a mixture of triflates, the desired of which, 7.15, isisolated by the appropriate chromatographic method. The fluoride isinstalled by treatment of 7.15 with tetra-n-butylammonium fluoride(TBAF) in an appropriate solvent, such as THF, yielding the desiredintermediate 7.16.

A specific compound of Formula 12 can be prepared as follows.

L-Thymidine 7.17, synthesized by Holy's method, is converted accordingto the literature procedures cited above to d4 nucleoside derivative7.18. Compound 7.18 is then treated with OsO₄ and NMO to givedihydroxylated product 7.19, which is triflated to provide 7.20(separated by silica gel chromatography from a mixture of itsregioisomers and di-triflated material). Compound 7.20 is then treatedwith TBAF to convert it to the desired compound 7.21. The diethylphosphonate may now be converted into any group that is desiredaccording to methods well known to chemists skilled in the art.

Examples 8-13 Synthesis of Representative Compounds of Formulae 13, 14and 15

Synthetic methodologies and intermediate compounds that can be used toprepare VX-148 analogs of formulae A, B, or C are described in Examples8-13.

Example 8 General Synthesis of Aniline Intermediate Useful for PreparingVX-148 Analogs of Formula A

A general scheme that is useful for converting a 3,5-difunctionalizednitrobenzene derivative to an aniline that can be used to prepare aVX-148 analog of the invention is illustrated above.

Example 9 Synthesis of Aniline Intermediate Useful for Preparing VX-148Analogs of Formula A

Representative compounds of the invention can be prepared as illustratedabove. 3-Hydroxy-5-nitro-benzoic acid is heated briefly in thionylchloride to generate the acid chloride. This is then condensed withO,N-dimethyl-hydroxylamine in the presence of a base such astriethylamine to produce the Weinreb amide which, upon reaction withmethyl lithium, gives the acetophenone derivative. This intermediate isthen treated with a base such as potassium carbonate in a dipolaraprotic solvent such as dimethylformamide, in the presence of an excessof E-1,4-dibromobutene. The monobromide is isolated by chromatographyand then subjected to treatment with triethylphosphite in a solvent suchas toluene (or other Arbuzov reaction conditions: see Engel, R.,Synthesis of carbon-phosphorus bonds, CRC press, 1988) to generate thedesired phosphonate diethyl ester. Thereafter, the carbonyl of theacetophenone is reduced enantioselectively using an appropriatehomochiral oxazaborolidine such as those described by Corey (J. Am.Chem. Soc., 1987, 109, 5551), and the resulting alcohol is displaced byazide using a method such as that described by Mitsunobu (Bull. Chem.Soc. Japan., 1971, 44, 3427). The azide is reduced to the amine underStaudinger conditions (Helv. Chim. Act., 1919, 2, 635) and protected asthe t-butyl carbonate. Finally, the desired aniline is generated by tin(II)-mediated reduction of the nitrobenzene. Using coupling reactionssimilar to those described in U.S. Pat. No. 6,054,472 and U.S. Pat. No.6,344,465 will give compounds of Formula A.

Example 10 Synthesis of VX-148 Analogs of Formula B

A general scheme that is useful for converting a 3,4-difunctionalizednitrobenzene derivative to an aniline, which can be converted to acompound of formula B using coupling reactions similar to thosedescribed in U.S. Pat. No. 6,054,472 and U.S. Pat. No. 6,344,465, isillustrated above.

Example 11 General Route to Representative Compounds of Formula C

Manipulation of a 3-substituted nitrobenzene 11.1 provides aniline 11.2,which can be converted to a compound of formula C using couplingreactions similar to those described in U.S. Pat. No. 6,054,472 and U.S.Pat. No. 6,344,465.

Example 12 General Route to Aniline Intermediate Useful For PreparingRepresentative Compounds of Formula C

3-Nitrobenzaldehyde reacts with a Grignard reagent to introduce a tetherbearing a protected alcohol and simultaneously to generate a benzylicalcohol, as shown. The alcohol is displaced by an azide in a mannersimilar to that described for Example 9. After deprotection, theliberated alcohol is alkylated with diethyl phosphonomethyltriflate(prepared according to Tetrahedron Lett. 1986, 27, 1477) using a basesuch as magnesium tert-butoxide in a solvent such as tetrahydrofuran.Subsequent transformations of the azide and nitro groups proceed in afashion similar to that described in Example 9. See Batt et al., Bioorg.Med. Chem. Lett. 1995, 5, 1549.

Example 13 General Route to Aniline Intermediate Useful For PreparingRepresentative Compounds of Formula C

3-tert-Butoxycarbonylamino-3-(3-nitro-phenyl)-propionic acid(commercially available) is coupled with 2-aminoethylphosphonic aciddiethyl ester (commercially available) using standard reagents for theformation of a secondary amide such as dicyclohexylcarbodiimide (DCC)and hydroxybenztriazole (HOBT), in a solvent such as dimethylformamide.Subsequent reduction of the nitro group proceeds in a fashion similar tothat described in the scheme in Example 9.

Example 14 General Route to Representative Compounds of Formula 16

The above scheme illustrates a general route that can be used to preparecompounds of Formula 16.

Example 15 Synthesis of Aniline Intermediate Useful for PreparingCompounds of Formula 16

Representative compounds of the invention can be prepared as illustratedabove. 3-Hydroxy-5-nitro-benzoic acid is heated briefly in acidicmethanol to generate the methyl ester. This is then treated with a basesuch as potassium carbonate in a dipolar aprotic solvent such asdimethylformamide, in the presence of an excess of E-1,4-dibromobutene.The monobromide is isolated by chromatography and then subjected totreatment with triethylphosphite in a solvent such as toluene (or otherArbuzov reaction conditions: see Engel, R., Synthesis ofcarbon-phosphorus bonds, CRC press, 1988) to generate the desiredphosphonate diethyl ester. Thereafter, the benzoate ester is saponifiedand reduced, and the resulting alcohol displaced by azide using a methodsuch as that described by Mitsunobu (Bull. Chem. Soc. Japan., 1971, 44,3427). The azide is reduced to the amine under Staudinger conditions(Helv. Chim. Acta, 1919, 2, 635) and protected as the t-butyl carbonate.Finally, the desired aniline is generated by tin (II)-mediated reductionof the nitrobenzene. The aniline is converted to a compound of Formula16 by the general procedures described in U.S. Pat. No. 6,054,472 andU.S. Pat. No. 6,344,465 as set forth in Example 10.

Example 16 General Route to Representative Compounds of Formula 17

Reagents suitable for use in the synthesis of representative compoundsof Formula 17 may be made by routes analogous to that shown in Example10, starting from 2-hydroxy-5-nitro-benzoic acid.

Example 17 General Route to Representative Compounds of Formula 18

Representative compounds of Formula 18 can be prepared as illustratedabove. The preparation of anilines of formula 17.2 is illustrated inExamples 11-13 above. Anilines of formula 17.2 can be converted tocompounds of formula 18 using procedures similar to those described inU.S. Pat. No. 6,054,472 and U.S. Pat. No. 6,344,465.

Example 18 Synthesis of Representative Compounds of Formula 19

Representative compounds of the invention can be prepared as illustratedabove. The phosphorus containing BILN-2061 analog 18.2 is synthesizedfrom the parent compound 18.1 by attachment of phosphorus containingmoiety to the carboxylic acid group. Compound 18.1, BILN-2061, isobtained by the procedure as described in WO 00/59929. The secondaryamine on the thiazole ring is protected with a suitable protectinggroup, such as Boc group before the formation of ester or amide as shownabove. The protected 18.1 is coupled with a phosphorus containing moietywith a hydroxy group by Mitsunobu reaction using triphenylphosphine anddiethyl azadicarboxylate, whereas an amide group is formed using aminogroup containing phosphonate reagent by suitable coupling reagents, suchas EDC-HOBt, BOP reagent etc. Deprotection of the coupled product givesthe desired phosphonate of type 18.2.

For instance, 18.1.1 is protected with Boc group using (Boc)₂O andtriethylamine and then treated with 2-hydroxyethylphosphonic aciddiethyl ester 18.7 in the presence of triphenylphosphine and diethylazadicarboxylate. The resulting ester is treated with trifluoroaceticacid to obtain analog 18.8, in which the linker is ethylene group.

Example 19 Synthesis of Representative Compounds of Formula 20

Synthesis of phosphonate analog of type 19.3 is illustrated above. Thephosphonate containing moiety is introduced to compound 19.1, or itsanalogs (R³=H, Me, Et, i-Pr), which are available by the proceduredescribed in WO 00/59929, by reductive amination using the phosphonatereagent 19.9 bearing aldehyde group.

For instance, compound 19.1.1 is treated with 2-oxoethyl phosphonic aciddiethyl ester 19.10 in the presence of sodium cyanoborohydride andacetic acid to provide compound 19.11, in which the linker is ethylene.

Example 20 Synthesis of Representative Compounds of Formula 21

Example 20 illustrates the preparation of compounds of type 20.4. Thesecondary amine on the thiazole ring and the carboxylic acid areprotected with suitable protecting groups. The methoxy group of thequinoline ring at 7-position is then demethylated using borontribromide. The phosphonate bearing moiety is then introduced on thishydroxy group in a suitable aprotic solvent such as, DMF, by treatingwith the phosphonate reagent 20.14, in the presence of a suitableorganic or inorganic base. In compounds 20.14, X is a leaving group suchas, but not limited to, bromide, chloride, iodide, p-toluenesulfonate,trifluoromethanesulfonate, or methanesulfonate.

The protecting groups are then removed to obtain the analog of type20.4.

For instance, the secondary amino group on the thiazole ring of 20.1 isprotected with Boc group using (Boc)₂O and triethylamine, and thecarboxylic acid is protected with t-butyl group using EDC, DMAP, andt-butyl alcohol, which result 20.15 as shown above. After the resulting20.15 is demethylated by boron tribromide, the alkylation of 20.16 withcesium carbonate and one equivalent of(trifluoromethanesulfonyloxy)methylphosphonic acid diethyl ester 17,followed by deprotection using trifluoroacetic acid gives 20.18, wherethe linkage is a methylene group as shown above.

Example 21 Synthesis of Representative Compounds of Formula 22

Synthesis of analogs 21.5 of BILN-2061 is illustrated above. Compound21.19 is synthesized by a procedure described in WO 00/59929 based onmethodology by T. Tsuda et al. (J. Am. Chem. Soc. 1980, 102, 6381).Compound 21.20, containing extra carbonyl group with suitable protectinggroups, R⁴ and R⁵, is synthesized by the procedure described for thesynthesis of 21.1 at WO 00/59929 using compound 21.19. The phosphonategroup bearing moiety is attached to the carbonyl group of 21.20 byreductive amination. The obtained secondary amine may be converted tothe tertiary amine by repeated reductive amination using formaldehyde oracetaldehyde to provide 21.5.2. The carbonyl group of 21.20 is alsoreduced to the corresponding alcohol and converted to phenyl carbonate,which is reacted with 21.21 to form compound 21.5.1, where the linkageis a carbamate. After the phosphonate bearing moiety is attached, theprotecting groups, R⁴ and R⁵, are removed by suitable methods well knowto those skilled in the chemical arts.

For instance, compound 21.22, obtained by the procedure of WO 00/59929,is treated with (Boc)₂O and triethylamine and followed by EDC, DMAP, andt-butyl alcohol to provide protected compound 21.23, as illustratedabove. Compound 21.23 is then treated with 2-aminoethylphosphonic aciddiethyl ester 21.24 in the presence of sodium cyanoborohydride andacetic acid, which results in the phosphonate bearing compound 21.25(R⁷=H), in which the phosphonate is attached to the structural corethrough a secondary amine, after deprotection by trifluoroacetic acid.Before deprotection, reductive amination of the secondary amine in thepresence CH₂O generates the tertiary amine, which is converted to 21.25(R⁷=Me) bearing a tertiary amine.

Example 22 Synthesis of Representative Compounds of Formula 23

Representative compounds of the invention can be prepared as illustratedabove. Condensation of commercially available 2-mercapto-ethanol andtrimethoxymethane (J. Org. Chem. USSR (Engl. Transl.) 1981, 1369-1371)generates heterocycle 22.3. Glycosidation using, for example,trimethylsilyl triflate and the phosphonate substituted alcohol 22.4,provides intermediate 22.5. Oxidation of sulfur to the sulfoxide usingmonoperoxyphthalic acid, magnesium salt (see U.S. Pat. No. 6,228,860col. 15 ln. 45-60) followed by a Pummerer rearrangement (see U.S. Pat.No. 6,228,860 col. 16 ln. 25-40) and base introduction (cytosine or5′-fluoro-cytosine) using conditions as outlined in U.S. Pat. No.6,228,860 (col. 17 ln. 15-42) provides the desired phosphonatesubstituted analogs 22.2.

Specifically, starting with heterocycle 22.3, using the above procedurebut using diethyl(hydroxymethyl)phosphonate 22.6, generates 22.7.Introduction of cytosine as outlined above provides the desired product22.8. Using the above procedure, but employing different phosphonatereagents 22.4 in place of 22.6, the corresponding products 22.2 bearingdifferent linking groups are obtained.

Example 23 Synthesis of Representative Compounds of Formula 24

Representative compounds of the invention can be prepared by reaction ofdOTC analogs of type 23.1 (obtained as described in U.S. Pat. No.6,228,860 col. 14 line 45 to col. 30 line 50 and references citedtherein) with the respective alkylating reagents 23.3. The above schemeshows the preparation of phosphonate linkage to dOTC through the 5′hydroxyl group. Substrate 23.1 (dOTC) is dissolved in a solvent such as,but not limited to, DMF, THF and is treated with a phosphonate reagentbearing a leaving group in the presence of a suitable organic orinorganic base. In compounds 23.3, Y is a leaving group such as, but notlimited to, bromide, chloride, iodide, p-toluenesulfonate,trifluoromethanesulfonate, or methanesulfonate.

For instance, 23.6 dissolved in DMF, is treated with one equivalent ofsodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 23.4, preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697, to givefluoro-cytosine phosphonate derivative 5, in which the linkage is amethylene group. Using the above procedure, but employing differentphosphonate reagents 23.3 in place of 23.4, the corresponding products23.2 bearing different linking groups are obtained.

Example 24 Synthesis of Representative Compounds of Formula 25

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs are prepared by reaction offuranoside purine nucleosides, structure 24.1 (obtained as described inU.S. Pat. No. 5,185,437 col. 9 ln. 16 to col. 35 In. 19 and referencescited therein) with the respective alkylating reagents 24.4. Illustratedabove is the preparation of the phosphonate linkage to furanosidenucleoside cores through the 5′-hydroxyl group. Parent analog 24.1 isdissolved in a solvent such as, but not limited to, DMF or THF, and istreated with a phosphonate reagent bearing a leaving group in thepresence of a suitable organic or inorganic base. In compounds 24.4, Xis a leaving group such as, but not limited to, bromide, chloride,iodide, p-toluenesulfonate, trifluoromethanesulfonate, ormethanesulfonate.

For instance, 24.5 (obtained as described in U.S. Pat. No. 5,185,437col. 9 ln. 16 to col. 35 ln. 19 and references cited therein) isdissolved in DMF, is treated with three equivalents of sodium hydrideand two equivalents of (toluene-4-sulfonylmethyl)-phosphonic aciddiethyl ester 24.6, prepared according to the procedures in J. Org.Chem. 1996, 61, 7697, to give the corresponding phosphonate 24.7, inwhich the linkage is a methylene group. Using the above procedure, butemploying different phosphonate reagents 24.4 in place of 24.6, thecorresponding products 24.2 bearing different linking groups areobtained.

Example 25 Synthesis of Representative Compounds of Formula 26

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 25.3 are prepared by reactingglycal 25.8 (obtained as described in J. Am. Chem. Soc. 1972, 94, 3213;in some cases the nucleoside bases may need prior protection) with therespective phosphonate alcohols 25.9, followed by treatment with iodinemonobromide (J. Org. Chem. 1991, 56, 2642-2647). Elimination of theresulting iodide followed by reduction with palladium on carbon providesthe desired product 25.3.

For instance, dihydrofuran 25.10 is dissolved in CH₂Cl₂ and is combinedwith 3.5 equivalents of diethyl(hydroxymethyl)phosphonate. The resultingsolution is treated with two equivalents of iodine monobromide at −25°C. The resulting phosphonate-iodide is treated with DBU and reducedunder hydrogenation conditions to afford the desired product 25.12.Using the above procedure, but employing different phosphonate reagents25.9 in place of 25.11, the corresponding products 25.3 bearingdifferent linking groups are obtained.

Example 26 Synthesis of Representative Compounds of Formula 27

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 26.2 are prepared by reactingglycal 26.3 (obtained as described in J. Am. Chem. Soc. 1972, 94, 3213)with phenylselenyl chloride followed by treatment with the respectivephosphonate alcohols 26.4 in the presence of silver perchlorate (J. Org.Chem. 1991, 56, 2642-2647). Oxidation of the resulting chloride usinghydrogen peroxide, followed by aminolysis treatment of uracil usingtriazole, 2-chlorophenyldichlorophosphate, pyridine and ammonia (Bioorg.Med. Chem. Lett. 1997, 7, 2567) and a palladium on carbon reductionprovides the desired product 26.2.

For instance, 26.3 dissolved in CH₂Cl₂, is treated with one equivalentof phenyl selenyl chloride at −70° C., followed by treatment with silverperchlorate in the presence of diethyl(hydroxymethyl) phosphonate togenerate selenide 26.7. The phosphonate is transformed into the d4CPanalog by first oxidation with hydrogen peroxide, followed by conversionof the uracil moiety to a cytosine, and finally hydrogenation to thedesired product 26.8. Using the above procedure, but employing differentphosphonate reagents 26.4 in place of 26.6, the corresponding products26.2 bearing different linking groups are obtained.

In some cases conversions to desired compounds may require the use ofsuitable protecting groups for the amino group of cytosine. Similarly,using different natural and unnatural bases with appropriate protectinggroups, other analogs containing a variety of bases can be prepared.

Example 27 Synthesis of Representative Compounds of Formula 28

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 27.2 are prepared by reaction ofddC 27.1 (D5782 Sigma-Aldrich, or prepares as described in J. Org. Chem.1967, 32, 817) with the respective alkylating reagents 27.3. The schemeshown above illustrates the preparation of phosphonate linkage to ddCthrough the 5′-hydroxyl group. Substrate 27.1 (ddC or an analog) isdissolved in a solvent such as, but not limited to, DMF or THF, and istreated with a phosphonate reagent bearing a leaving group, in thepresence of a suitable organic or inorganic base. In compounds 27.3, Xis a leaving group such as, but not limited to, bromide, chloride,iodide, p-toluenesulfonate, trifluoromethanesulfonate, ormethanesulfonate.

For instance, 27.1 dissolved in DMF, is treated with two equivalent ofsodium hydride and two equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 27.4, preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697, to give ddCphosphonate 27.5 in which the linkage is a methylene group. Using theabove procedure, but employing different phosphonate reagents 27.3 inplace of 27.4, the corresponding products 27.2 bearing different linkinggroups are obtained.

Example 28 Synthesis of Representative Compounds of Formula 29

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 28.2 are prepared by reaction ofdioxolanyl purine nucleosides, structure 28.1 (obtained as described inU.S. Pat. No. 5,925,643 col. 4 ln. 47 to col. 12 ln. 20 and referencestherein) with the respective alkylating reagents 28.3. Illustrated aboveis the preparation of phosphonate linkage to dioxalane nucleoside coresthrough the 5′-hydroxyl group. Parent analog 28.1 is dissolved in asolvent such as, but not limited to, DMF and/or THF, and is treated witha phosphonate reagent bearing a leaving group, in the presence of asuitable organic or inorganic base. In compounds 28.3, X is a leavinggroup such as, but not limited to, bromide, chloride, iodide,p-toluenesulfonate, trifluoromethanesulfonate, or methanesulfonate.

For instance, 28.4 dissolved in DMF, is treated with five equivalents ofsodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 28.5, preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697, to give thecorresponding phosphonate 28.6, in which the linkage is a methylenegroup. Using the above procedure, but employing different phosphonatereagents 28.3 in place of 28.5, the corresponding products 28.2 bearingdifferent linking groups are obtained.

Example 29 Synthesis of Representative Compounds of Formula 30

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 29.2 are prepared by reaction of3TC (29.1). (obtained as described in U.S. Pat. No. 5,047,407 col. 9line 7 to col. 12 line 30 and references cited therein) with therespective alkylating reagents 29.3. Illustrated above is thepreparation of phosphonate linkage to 3TC through the 5′-hydroxyl group.3TC is dissolved in a solvent such as, but not limited to, DMF and/orTHF, and is treated with a phosphonate reagent bearing a leaving group,in the presence of a suitable organic or inorganic base. In compounds29.3, X is a leaving group such as, but not limited to, bromide,chloride, iodide, p-toluenesulfonate, trifluoromethanesulfonate, ormethanesulfonate.

For instance, 29.1 dissolved in DMF, is treated with one equivalent ofsodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 29.4 (preparedaccording to the procedure in J. Org. Chem. 1996, 61, 7697) to give 3TCphosphonate 29.5, in which the linkage is a methylene group. Using theabove procedure, but employing different phosphonate reagents 29.3 inplace of 29.4, the corresponding products 29.2 bearing different linkinggroups are obtained.

Example 30 Synthesis of Representative Compounds of Formula 31

Representative compounds of the invention can be prepared as illustratedabove. Starting with the known oxathiolan-5-one (30.3) (Acta Chem.Scand., Ser. A 1976, 30, 457), reduction followed by base introductionusing the conditions outlined in U.S. Pat. No. 5,914,331 (col. 11 ln. 62to col. 12 ln. 54) provides the substrate for the Pummerer reaction.Oxidation using m-chloroperbenzoic acid in methanol (U.S. Pat. No.5,047,407 col. 12 ln. 35 to col. 12 ln. 50) generates sulfoxide 30.4.The Pummerer reaction in the presence of the phosphonate linked alcohol30.5 and acetic anhydride provides phosphonate 30.6.

As an example, subjecting oxathiolan-5-one to conditions above but using5-fluoro-2-[(trimethylsilyl)oxy]-4-pyrimidinamine followed by oxidationprovides intermediate 30.7. Introduction of phosphonate moiety 30.8,using Pummerer conditions (Org. React. 1991, 40, 157) provides thediethyl phosphonate product 30.9.

Example 31 Synthesis of Representative Compounds of Formula 32

Representative compounds of the invention can be prepared as illustratedabove. Alcohol 31.3 can be prepared as described in J. Chem. Soc.,Perkin Trans. 11994, 1477. Note that other base derivatives can beprepared in a similar manner starting with their respective bases.Displacement of the chloride of 31.3 with an amine in ethanol underreflux conditions (U.S. Pat. No. 5,034,394, col. 9, ln. 60 to col. 10ln. 21) provides the key intermediate alcohol. Treatment of this alcoholwith the respective alkylating reagents 31.4, provides the desiredphosphonate substituted analogs 31.2. In the above compounds, R₆ is H,R₇ is cyclopropyl, R₃ is NH₂.

As an example, treatment of the key intermediate alcohol, as describedabove (J. Chem. Soc., Perkin Trans. 1994, 1, 1477), with one equivalentof sodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 31.6 (preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697) affords ABCphosphonate 31.7, in which the linkage is a methylene group. Using theabove procedure, but employing different R₃, R₆, R₇ and phosphonatereagents 31.4 in place of 31.6, the corresponding products 31.2 bearingdifferent linking groups are obtained.

Example 32 Synthesis of Representative Compounds of Formula 33

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 32.5 are prepared by reaction of32.3 (for example, AZT (A 2169, Sigma Aldrich or obtained as describedin U.S. Pat. No. 4,724,232) or 3′-deoxythymidine (D 1138 Sigma Aldrich))with the respective alkylating reagents 32.4. Further modification ofeither the base or the 3′-substituent can be carried out as illustratedabove. AZT is dissolved in a solvent such as, but not limited to, DMFand/or THF, and is treated with a phosphonate reagent bearing a leavinggroup, in the presence of a suitable organic or inorganic base. Incompounds 32.4, X is a leaving group such as, but not limited to,bromide, chloride, iodide, p-toluenesulfonate,trifluoromethanesulfonate, or methanesulfonate.

Treatment of compound 32.5 with methyl hypobromite provides the5-bromo-6-alkoxy analog 32.6 (J. Med. Chem. 1994, 37, 4297 and U.S.patent Ser. No. 00/22600). Compound 32.6 can be elaborated by reducingthe 3′-azide to the amine and converting the amine to the correspondingacetyl to provide compounds 32.7.

For instance, 32.1 dissolved in DMF, is treated with one equivalent ofsodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 32.8 (preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697) to give AZTphosphonate 32.9, in which the linkage is a methylene group. Treatmentwith methyl hypobromite followed by hydrogenation provides analog 32.10.Using the above procedure, but employing different phosphonate reagents32.4 in place of 32.8, the corresponding products 32.2 bearing differentlinking groups are obtained. Additionally, the R₃-R₅ groups can bevaried to generate other compounds.

Example 33 Synthesis of Representative Compounds of Formula 34

Representative compounds of the invention can be prepared as illustratedabove. Starting with commercially available glycidol, silyl protectionof the alcohol followed by a lithium-mediated opening of the epoxidegenerates alcohol 33.4 (see Angew. Chem., Int. Ed. Engl. 1998, 37,187-192). Introduction of the appropriately protected bases usingMitsunobu reaction conditions (Tetrahedron Lett. 1997, 38, 4037-4038;Tetrahedron 1996, 52, 13655) followed by acid mediated removal of thesilyl protecting group (J. Org. Chem. 1980, 45, 4797) and dithianeremoval and in situ cyclization (J. Am. Chem. Soc. 1990, 112, 5583)produces furanoside 33.5. Introduction of phosphonate linkage using theappropriate alcohol in the presence of TMSOTf (Synlett 1998, 177)generates analog 33.2.

For instance, 3 equivalents of DIAD (in 3 portions) is added dropwise toa stirred solution of alcohol 33.4 and adenine (3 equivalents) indioxane. The reaction is stirred for 20 hours. The resulting product istreated with hydrochloric acid in ethanol for 15 hours and filtered. Theresidue is stirred with [bis(trifluoroacetoxy)iodo]benzene (1.5equivalents) in methanol to generate 33.7. Lewis acid-mediated reaction(Synlett 1998, 177) of diisopropyl hydroxymethylphosphonate 33.8(Tetrahedron Lett. 1986, 27, 1477) produces a diastereomeric mixture ofphosphonates 33.9, in which the linkage is a methylene group. Using theabove procedure, but employing different appropriately protected basesand phosphonate reagents 33.6 in place of 33.8, the correspondingproducts 33.2 bearing different linking groups are obtained.

Example 34 Synthesis of Representative Compounds of Formula 35

Representative compounds of the invention can be prepared as illustratedabove. Phosphonate substituted analogs 34.2 are prepared by reaction ofFTC (34.1) (obtained as described in U.S. Pat. No. 5,914,331 col. 10line 40 to col. 18 line 15 and references cited therein) with therespective alkylating reagents 34.3. Illustrated above is thepreparation of phosphonate linkage to FTC through the 5′-hydroxyl group.FTC is dissolved in a solvent such as, but not limited to, DMF and/orTHF, and is treated with a phosphonate reagent bearing a leaving group,in the presence of a suitable organic or inorganic base. In compounds34.3, X is a leaving group such as, but not limited to, bromide,chloride, iodide, p-toluenesulfonate, trifluoromethanesulfonate, ormethanesulfonate.

For instance, 34.1 dissolved in DMF, is treated with one equivalent ofsodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 34.4 (preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697) to give FTCphosphonate 34.5 in which the linkage is a methylene group. Using theabove procedure but employing different phosphonate reagents 34.3 inplace of 34.4, the corresponding products 34.2 bearing different linkinggroups can be obtained.

Example 35 Synthesis of Representative Compounds of Formula 37

Representative compounds of the invention can be prepared as illustratedabove. The synthetic scheme above outlines a sequence for producing aphosphonate linked HCV polymerase inhibitor. Compound 35.1 (WO 02/04425page 40, paragraph 1, Example 2) is reacted with amino acid 35.2(purchased from a supplier such as Sigma-Aldrich) under standard peptidecoupling conditions: 35.1 is combined with, for example, HATU, HOAT, 2equivalents of EtNiPr2, in NMP, DMF, THF, CH₂Cl₂, or DMA for 2 to 60minutes at room temperature. The resulting mixture is then added to 35.2for 10 minutes to 5 days at room temperature to 100° C. After standardwork-up and purification procedures, compound 35.3 is reacted with 2equivalents of NaH in anhydrous DMF (NMP, DMSO, or THF) followed by theaddition of 35.4 produce substituted phosphonate 35.5 after work-up andpurification.

In an analogous fashion, 35.3 can be converted to 35.9, 35.10, and 35.11as illustrated above. The R₁ groups attached to the phosphonate esters35.9, 35.10 and 35.11 may be changed using established chemicaltransformations well know to those of skill in the chemical arts.

Example 36 Synthesis of Representative Compounds of Formula 38

Representative compounds of the invention can be prepared as illustratedabove phosphonate 36.14 is produced from 36.12 (WO 02/04425, page 59,paragraph 1) by the action of HATU/HOAT/EtN(i-Pr)₂ on 36.12 and 36.13using protocols familiar to one skilled in the art in a polar aproticsolvent such as, but not limited to, NMP, DMF, THF, and DMSO.

Synthetic methodologies and intermediate compounds that can be used toprepare analogs of Formulae 39-43 are described below in Examples 37-40.

Example 37 General Route to Representative Compounds of Formula 39

This Example describes linkage groups (Linker) that can be used tocovalently attach a phosphonate containing group to the compoundsdescribed in patent EP1162196A1. This Example also illustrates thechemical reactions that can be used to attach the linker-phosphonatemoiety to parent compounds.

Representative compounds of the invention can be prepared as illustratedabove. Compound 37.2 in an appropriate aprotic solvent can be treatedwith at least two equivalents of an appropriate organic or inorganicbase. An appropriate electrophile bearing a leaving group, such as, butnot limited to, diisopropyl bromomethylphosphonate, is added to producecompound 37.3.

Suitable aprotic solvents include, but are not limited to, dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidinone. Suitableorganic or inorganic base include, but are not limited to, sodiumhydride, potassium carbonate, and triethylamine. Suitable leaving groupsinclude, but are not limited to, chlorine, bromine, iodine,p-toluenesulfonate, methanesulfonate, and trifluoromethanesulfonate.

Linker Type I: alkylation of the parent compound 37.2 with anelectrophile containing phosphonate. At least five types of functionalgroups can be directly alkylated. The functional groups include, but arenot limited to, hydroxyl, amine, amide, sulfonamide, and carboxylicacid. Some representative examples of compounds from JT patentEP1162196A1 bearing one of the above groups are listed in the followingtable.

Group Example # in JT Patent EP1162196A1 Hydroxyl 3, 18, 28, 52, 107,1005, 1026, 1012, 1021, 288, 289, 294, 2231 Amine 22, 39, 308, 152, 160,161 Amide 1260, 1263, 1270, 130, 1280, 162, 190, 195, 205, 225, 1388,262, 268, 316 Sulfonamide 41, 42, 57, 1357, 249, 250, 283, 285Carboxylic acid 246, 140, 141, 184, 189, 1015, 1020, 1054, 1069, 1084,255, 260, 292, 293

Example 38 General Route to Representative Compounds of Formulae 40 and41

This Example describes linkage groups (Linker) that can be used tocovalently attach a phosphonate containing group to the compoundsdescribed in patent EP1162196A1. This Example also illustrates thechemical reactions that can be used to attach the linker-phosphonatemoiety to parent compounds.

Representative compounds of the invention can be prepared as illustratedabove. Compound 38.4 in an appropriate solvent can be treated with asuitable olefin containing a phosphonate group, catalytic amount of apalladium catalyst, with an optional phosphine ligand, and at least twoequivalents of an appropriate base to produce 38.5. Other optionalphosphine ligands may be used according to procedures know to those ofskill in the art (see Encyclopedia of Reagents for Organic Synthesis,Leo A. Paquette Ed.-in-Chief, John Wiley & Sons, Chichester, UK, 1995).

Suitable solvents include, but are not limited to dimethyl formamide,dimethylacetamide, and N-methylpyrrolidinone. Suitable palladiumcatalysts include, but are not limited to palladium acetate, palladiumchloride, and palladium bis(triphenylphosphine) “dichloride. Suitablephosphine ligands include, but are not limited to triphenylphosphine,and tri(o-toluoyl)phosphine. Suitable bases include, but are not limitedto triethylamine, diisopropylethylamine, and tributylamine. Suitableolefins containing a phosphonate group include, but are not limited toesters of vinylphosphonic acid and allylphosphonic acid.

Linker Type II: coupling between an aryl bromide in a parent compoundand an olefin containing a phosphonate group. Some representativeexamples of compounds bearing an aryl bromide are Examples. 1, 2, 4, 7,244, 180, 1023, 1086, 1087, 1093, 1208, 1220, and 1301, as listed in JTpatent EP1162196A1.

Linker Type III: hydrogenation of Linker Type II.

The following is an example illustrating hydrogenation of Linker TypeII.

Compound 38.5 is treated with potassium azodicarboxylate, acetic acidand pyridine (as described in Liebigs Ann. Chem. 1984; 98-107) to give38.6. In cases where the parent structure is stable to hydrogenationusing palladium catalyst or Raney nickel, those combination of reagentscan also be used to effect the same transformation. Common solvents usedfor those procedures include, but are not limited to, methanol, ethanol,isopropanol, and acetic acid.

Example 39 General Route to Representative Compounds of Formulae 42 and43

The following scheme illustrates the reductive amination reaction.

Representative compounds of the invention can be prepared as illustratedabove. Ketone 39.7 in an appropriate solvent can be treated with asuitable amine containing a phosphonate group, and an appropriatereducing agent to produce amine 39.8.

Linker Type IV: Reductive Amination.

Variation 1: coupling between an amine containing a phosphonate groupand a parent compound containing an aldehyde or a ketone group. Somerepresentative examples of parent compounds bearing an aldehyde or aketone group are Examples 36, 1234, 1252, 1256, and 2241 as listed in JTpatent EP1162196A1.

Variation 2: Coupling between an aldehyde or ketone containing aphosphonate group and a parent compound containing an amine group. Somerepresentative examples of parent compounds bearing an amino group areExamples 22, 39, 308, 152, 160, and 161 as listed in the JT patentEP1162196A1.

The following is an example illustrating a Variation 2-type of reductiveamination reaction.

Aryl amine 39.9 in an appropriate solvent can be treated with a suitablealdehyde or a ketone containing a phosphonate group, and an appropriatereducing agent to produce secondary amine 39.10.

In the above two variations of Linker Type IV, the appropriate reducingagents include, but are not limited to, sodium borohydride, sodiumcyanoborohydride, and sodium triacetoxyborohydride. The appropriatesolvents include, but are not limited to, methanol, ethanol, and1,2-dichloromethane.

Example 40 General Route to Representative Compounds of Formulae 44 and45

The following is an example illustrating an amide formation reaction.

Representative compounds of the invention can be prepared as illustratedabove. Aryl amine 40.11 in an appropriate solvent can be treated with acarboxylic acid containing a phosphonate group and an appropriatecoupling reagent to give amide 40.12.

Linker Type V: Amide Formation

Variation 1: amide formation between a carboxylic acid containing aphosphonate group and a parent compound containing an amine group. Somerepresentative examples of parent compounds bearing an amino group areExamples 22, 39, 308, 152, 160, and 161 as listed in JT patentEP1162196A1.

Variation 2: amide formation between an amine containing a phosphonategroup and a parent compound containing a carboxylic acid. Somerepresentative examples of parent compounds bearing a carboxylic acidare Examples 246, 184, 189, 1015, 1020, 1054, 255, 260, 292, 295, 305,and 2013 as listed in JT patent EP1162196A1.

The following is an example illustrating this type of amide formationreaction.

Representative compounds of the invention can be prepared as illustratedabove. Compound 40.13 in an appropriate solvent can be treated with anamine containing a phosphonate group and an appropriate coupling reagentto give 40.14.

In the above two variations of Linker Type 5, the appropriate couplingreagents include, but are not limited to, DCC and EDC. The appropriatesolvents include, but are not limited to, dimethylformamide anddichloromethane.

Example 41 Synthesis of Representative Compounds of Formula 46

Representative compounds of the invention can be prepared as illustratedabove. The 5′-hydroxyl group of ribavirin (41.2) can be selectivelyprotected with an appropriate protecting group. The product, 41.3, canbe treated with benzoyl chloride, an appropriate base, in the presenceof catalytic amount of 4-dimethylaminopyridine, to convert 2′- and3′-hydroxyl groups to their corresponding benzoyl esters, producingdibenzoate 41.4. The 5′-hydroxyl group can be selectively deprotected toafford alcohol 41.5. Following procedure described for the analogouscompound in U.S. Pat. No. 6,087,482, FIG. 2, dibenzoate 41.4 can beconverted to 41.7 in a three-step sequence. Treating electrophile 41.7with a coupling agent, such as trimethylsilyl trifluoromethanesulfonate,in the presence of an appropriate alcohol containing a phosphonate groupcan produce phosphonate 41.8. Treating 41.8 with aqueous sodiumhydroxide can deprotect the 2′- and 3′-hydroxyl groups to provide diol41.1. Note that R^(P1) and R^(P2) in 41.8 and 41.1 can be the same ordifferent protecting groups.

Example 42 Synthesis of Representative Compounds of Formula 47

Representative compounds of the invention can be prepared as illustratedabove. The synthesis of3-cyano-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,2,4-triazole (42.2)is described in US 2002/0156030 A1, page 6, paragraph 0078 to paragraph0079. Using this starting material, one can synthesize compound 42.1using the sequence of chemical transformations outlined above.

Appropriate protection and deportection procedures (see Greene and Wuts,Protective Groups in Organic Synthesis, 1999) can be employed to prepare42.3, in which the 5′-hydroxyl group is protected, while the 2′-, and3′-hydroxyl groups are not. Subsequent protection, deprotectionprocedures can introduce protecting groups such as benzoyl group to the2′- and 3′-hydroxyls, leaving the 5′-hydroxyl group unprotected as inalcohol 42.4. Oxidation can convert the primary alcohol in 42.4 to thecorresponding carboxylic acid or its ester. An optional deprotection ofthe ester can give the acid 42.5 as product. Further oxidation usingoxidant such as lead tetraacetate can convert acid 42.5 to electrophile42.6, in which the leaving group is an acetate. Treating 42.6 with analcohol containing a phosphonate moiety in the presence of appropriatecoupling agent, such as trimethylsilyl trifluoromethanesulfonate,affords phosphonate 42.8. Finally, treating 42.8 with the proceduredescribed in US 2002/0156030 A1, page 6, paragraph 0081, providesphosphonate 42.1. Note that R^(P1) and R^(P2) in 42.7, 42.8 and 42.1 donot need to be the same.

Example 43 Synthesis of Representative Compounds of Formula 48

Representative compounds of the invention can be prepared as illustratedabove. Compound 43.2 can be prepared from 43.1 by a series of selectiveprotections of the 2′-, 3′-, and 5′-hydroxyl groups to give 43.6. The5′-hydroxyl can then be selectively deprotected to give alcohol 43.7.Compound 43.7 in an appropriate aprotic solvent can be treated with atleast two equivalents of an appropriate organic or inorganic base, andan appropriate electrophile bearing a leaving group, as in the structureX-linker-POR^(P1)R^(P2), where X is a leaving group, to producephosphonate 43.8. Appropriate deprotection procedures can be employed toconvert 43.8 to diol 43.2. Note that R^(P1) and R^(P2) in 43.8 and 43.2do not need to be the same.

Suitable aprotic solvents include, but are not limited to dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidinone. Suitableorganic or inorganic base include, but are not limited to sodiumhydride, potassium t-butoxide, and triethylamine. Suitable leavinggroups include, but are not limited to, chlorine, bromine, iodine,p-toluenesulfonate, methanesulfonate, and trifluoromethanesulfonate.

Example 44 Synthesis of Representative Compounds of Formulae 49 and 50

Representative compounds of the invention can be prepared as illustratedabove. Triol 44.1 can be converted to alcohol 44.9, having anunprotected 2′-hydroxyl, by selecting appropriate protecting groups forthe 3′- and 5′-hydroxyl groups through a series of protection anddeprotection sequences. Alkylation of 44.9 by an electrophile containinga phosphonate as described in Example 43 can produce 44.10. Afterappropriate deprotection, 44.10 can be converted to phosphonate 44.3.

The preparation of 44.4 is illustrated above. The reaction sequence andconditions are similar to that of 44.2 and 44.3 described above.

Representative compounds of the invention can also be prepared byfollowing the sequences illustrated in Examples 41-44 using enantiomericstarting materials corresponding to, for example, compounds 41.2 and42.2 to provide compounds of Formulae 51 and 52, respectively.

Example 45 Synthesis of Representative Compounds of Formula 53

Representative compounds of the invention can be prepared as illustratedabove. In J. Carbohydrate Res. 1989, 163, the synthesis of azide 45.6was fully described using L-sorbose as starting material. Preparation ofphosphonate 45.4 is outlined above. Selective protection of the primaryalcohol in 45.6 gives alcohol 45.7 (see Greene and Wuts, ProtectiveGroups in Organic Synthesis, 1999). Compound 45.7 in an appropriateaprotic solvent is treated with at least one equivalents of anappropriate organic or inorganic base. Addition of an appropriateelectrophile bearing a leaving group affords compound 45.8.

Transformation of 45.8 to 45.10 is exemplified in J. Carbohydrate Res.1989, 163. R¹ can be introduced to the amine nitrogen using analkylation or reductive amination procedure. Final deprotection of theprimary alcohol affords phosphonate 45.4. Note that R^(P1) and R^(P2) inthe sequence from 45.8 to 45.4 do not need to be the same.

Suitable aprotic solvents include, but are not limited to dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidinone. Suitableorganic or inorganic base include, but are not limited to sodiumhydride, potassium carbonate, and triethylamine. Suitable leaving groupsinclude, but are not limited to, chlorine, bromine, iodine,p-toluenesulfonate, methanesulfonate, and trifluoromethanesulfonate.

Example 46 Synthesis of Representative Compounds of Formula 53

Representative compounds of the invention can be prepared as illustratedabove. The details of several individual steps are described above inExample 45. Group R⁴ is a cyclic protecting group that protects the1,2-diol in 46.16. Note that R¹¹ and R¹² in 46.17 and 46.2 do not needto be the same.

Example 47 Synthesis of Representative Compounds of Formula 54

Representative compounds of the invention can be prepared as illustratedabove. The preparation of 47.3 is outlined above. The details ofindividual steps are described in Example 45. Note that R^(P1) andR^(P2) in 47.18, 47.19 and 47.3 do not need to be the same.

Example 48 Synthesis of Representative Compounds of Formula 56

Representative compounds of the invention can be prepared as illustratedabove. The details of each individual step are outlined in Example 45.Note that R^(P1) and R^(P2) in 48.23 and 48.5 do not need to be thesame.

Example 49 Synthesis of Representative Compounds of Formula 57

Representative compounds of the invention can be prepared as illustratedabove. The preparation of 49.5 is described in WO 92/15582 A1 page 18line 30 to page 19 line 14. Compound 49.5 in an appropriate aproticsolvent can be treated with at least one equivalent of an appropriateorganic or inorganic base. An appropriate electrophile bearing a leavinggroup (X) containing a phosphonate, such as diisopropylbromomethylphosphonate, is added to produce compound 49.6.

Suitable aprotic solvents include, but are not limited to, dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidinone. Suitableorganic or inorganic base include, but are not limited to, sodiumhydride, potassium carbonate, and triethylamine. Suitable leaving groupsinclude, but are not limited to, chlorine, bromine, iodine,p-toluenesulfonate, methanesulfonate, and trifluoromethanesulfonate.

Example 50 Synthesis of Representative Compounds of Formula 58

The preparation of 50.7 is described in WO 92/15582 A1 page 22 line 28to page 23 line 3. Compound 50.7 in an appropriate solvent can betreated with a suitable amine containing a phosphonate group, and anappropriate base to produce phosphonate 50.8. Suitable bases include,but are not limited to, N-methylmorpholine, diisopropylethylamine, andpotassium carbonate. Suitable aprotic solvents include, but are notlimited to, DMF, DMPU, and NMP.

Example 51 Synthesis of Representative Compounds of Formula 59

Representative compounds of the invention can be prepared as illustratedabove. The amino group in 51.1 can be protected with an appropriateprotecting group to give 51.9. Compound 51.9 in an appropriate aproticsolvent can be treated with at least one equivalent of an appropriateorganic or inorganic base. An appropriate electrophile bearing a leavinggroup (X) containing a phosphonate, such as diisopropylbromomethylphosphonate, is added to produce compound 51.10. Appropriatedeprotection procedure will convert 51.10 to 51.11.

Suitable aprotic solvents include, but are not limited to, dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidinone. Suitableorganic or inorganic base include, but are not limited to sodiumhydride, potassium carbonate, and triethylamine. Suitable leaving groupsinclude, but are not limited to, chlorine, bromine, iodine,p-toluenesulfonate, methanesulfonate, and trifluoromethanesulfonate.

Example 52 Synthesis of Representative Compounds of Formula 60

Representative compounds of the invention can be prepared as illustratedabove. The phosphorous-containing compounds can be readily preparedaccording to conventional synthetic procedures where aphosphonate-containing group may be attached to the starting compoundusing established chemical processes. The preparations may be carriedout from the precursor compounds described in WO 99/62513. The methodsfor these conversions from precursor compounds to thephosphorous-containing compounds are described in Comprehensive OrganicTransformations, Richard C. Larock, ed, VCH, 1989; Comprehensive OrganicSynthesis, Barry M. Trost and Ian Fleming eds., Pergamon Press, 1991.Protection of functional groups during the transformations is describedin Protective Groups in Organic Synthesis, Theodora W. Greene and PeterG. M. Wuts, eds., Wiley, 1999.

Examples 53-58 illustrate syntheses of Formula 61. Thephosphorous-containing compounds of the present invention with formula61 can be readily prepared according to the conventional syntheticprocedures exemplified in Examples 53-58. The preparations may becarried out from the precursor compounds described in WO 00/39085, WO00/75122, WO 01/00578 and WO 01/95905. The methods for these conversionsfrom precursor compounds to the phosphorous-containing compounds aredescribed in Comprehensive Organic Transformations, Richard C. Larocked., VCH, 1989; Comprehensive Organic Synthesis, Barry M. Trost and IanFleming eds., Pergamon Press, 1991. Protection of function groups duringthe transformation is described in Protective Groups in OrganicSynthesis, Theodora W. Greene and Peter G. M. Wuts, eds., Wiley, 1999.For Examples 53-55: In one embodiment of the invention, Z can be carbon;in another embodiment of the invention, Z can be nitrogen.

Example 53 General Route to Representative Compounds of Formula 61

Representative compounds of the invention can be prepared as illustratedabove. Compounds 53.1 and 53.2 can be converted to compound 53.3 undercondensation condition in the presence of the base such as lithiumbis(trimethylsily)amide (WO 00/75122).

Example 54 General Route to Representative Compounds of Formula 61

Representative compounds of the invention can be prepared as illustratedabove. Prop-2-enone derivative 54.1 can be protected as a silyl ethersuch as a TBS ether, as in 54.2. Alkylation of silyl-protected 54.2 withphosphonate 54.3, where LG is OTf, Br, or Cl, is accomplished accordingto methods described in J. Heterocyclic Chem. 1995, 32, 1043-1050 andBioorg. Med. Chem. Lett. 1999, 9, 3075-3080. Phosphonate 54.5 isobtained upon removal of the silyl protection group of 54.4, using asuitable reagent such as tetrabutylammonium fluoride (TBAF).

Example 55 General Route to Representative Compounds of Formula 61

Representative compounds of the invention can be prepared as illustratedabove. A carbamate moiety can be used to connect hydroxy-phosphorouscompound 55.2 to heterocycle 55.1. Commonly used reagents such as CDIcan are useful in such transformations. Removal of the TBS group of 55.3provides phosphonate 55.4.

A urea moiety can be used to connect hydroxy-phosphorous compound 55.6to heterocycle 55.5. Commonly used reagents such as CDI can are usefulin such transformations. Removal of the TBS group of 55.7 providesphosphonate 55.8.

Example 56 General Route to Representative Compounds of Formula 61

Representative compounds of the invention can be prepared as illustratedabove.

Example 57 Synthesis of Representative Compounds of Formula 61

Representative compounds of the invention can be prepared as illustratedabove.

Example 58 Synthesis of Representative Compounds of Formula 61

Representative compounds of the invention can be prepared as illustratedabove.

Examples 59 and 60

Synthetic methodologies and intermediate compounds that can be used toprepare analogs of formulae D, E, F and G are described in Examples 59and 60. These compounds are representative examples of compounds ofFormulae 62-65.

Example 59 Synthesis of Representative Compounds of Formulae 62 and

Representative compounds of the invention can be prepared as illustratedabove. Betulinic Acid (59.1.1) may be purchased from Sigma (Cat. No.85505-7) or alternatively isolated from the stem bark of Ziziphusmauriitiana Lam. (Rhamnaceae) as described in WO 02/16395 A1.Dihydrobetulinic acid is obtained by hydrogenation of betulinic acid asdescribed in WO 02/16395 A1. Example 59 illustrates the synthesis ofbetulinic and dihydrobetulinic derivatives D and F.

The hydroxyl group of betulinic acid 59.1.1 is first protected with asuitable protecting group, such as benzyl ether as described in Greeneand Wuts, Protecting Groups in Organic Synthesis, third edition, JohnWiley and Sons, Inc. The protected hydroxyl is then converted into theacid chloride 59.2 which is then treated with an amine phosphonic acidof the general formula 59.3 to form the amide 59.4. Deprotection of thehydroxyl gives products of the general formula 59.1. Dihydrobetulinicacid can be treated in exactly the same manner as 59.1 to give productsof the general formula F.

For instance, betulinic acid is dissolved in a suitable solvent such as,DMF and treated with 2 equivalents of benzyl bromide along with anappropriate base such as NaH. The undesired benzyl protected carboxylateis removed by treatment with aqueous K₂CO₃ to give the benzyl etherwhich is then treated with 4 equivalents of oxalyl chloride in asuitable solvent, such as chloroform or methylene chloride (as describedin J. Med. Chem. 2002, 45, 4271-4275) to give 59.5. Acid chloride 59.5is then treated with 2 equivalents of diethyl 2-aminoethyl-1-phosphonate59.6 (prepared as described in J. Med. Chem. 1998, 41, 4439-4452), alongwith 4 equivalents of a tertiary amine such as, for example,triethylamine to give the amide 59.7. Final deprotection of the benzylether is achieved by treatment with lithium di-tert-butylbiphenyl in THFat −78° (as described in J. Am. Chem. Soc. 1991, 113, 8791-8796)followed by purification using reverse-phase or normal phasechromatography to give 59.8. Using the above procedure, but employingdifferent phosphonate reagents 59.3 in the place of 59.6, thecorresponding products of general type D and F bearing different linkinggroups can be prepared.

Example 60 Synthesis of Representative Compounds of Formulae 63 and 65

Representative compounds of the invention can be prepared as illustratedabove. Example 60 describes the synthesis of phosphonate derivatives ofbetulinic and dihydrobetulinic acid of the general formula E and G.Betulinic Acid 60.1 is treated with, for example, an appropriatelysubstituted acid chloride (as described in Tetrahedron Lett. 1997, 38,4277-4280) to form halide derivative 60.9. Halide 60.9 is then treatedwith a hydroxylphosphonic acid of the general formula 60.10 along with asuitable base to give compound 60.11. Dihydrobetulinic acid can betreated in exactly the same manner as 60.1 to give products of thegeneral formula G.

For instance, 60.1 is dissolved in a suitable solvent such as THF,treated with 2 equivalents of a suitable base, such as triethylamine,and 2 equivalents of bromoacetyl chloride to give compound 60.12. Thebromo derivative 60.12 is then treated with diethylhydroxymethylphosphonate (which can be purchased from Sigma (Cat. No.39262-6)) along with a suitable base, such as Cs₂CO₃ (as described inTetrahedron Lett. 1999, 40, 1843-1846) to give compound 60.14.Dihydrobetulinic acid can be treated in exactly the same manner to giveproducts of the general formula G. Using the above procedure, butemploying different phosphonate reagents 60.10 in the place of 60.13,the corresponding products E and G bearing different linking groups canbe prepared.

Examples 61-63

The analogs described in Examples 61-63 show a methylene group as thelinker. The linker may, however, be any other group described in thisspecification.

Example 61 General Route to Representative Compounds of Formula 66Cyclobutanes:

4-Membered Ring Nucleoside Series:

Representative compounds of the invention can be prepared as illustratedabove. Compound 61.1 (Base=G), described in the literature, was shown tohave reasonable anti-HIV activity (50-100 μM, cf. lubocavir 30 μM).Therefore, one target is its isosteric phosphonate derivative 61.2.Also, compound 61.1 can be derivatized to its phosphonate 61.4. In asimilar manner, one may add a phosphonate group onto lubocavir toprepare carbocyclic 61.5, or alternatively carbocyclic isostere 61.6.Compounds 61.6 have a hydroxyl group analogous to the 3′-hydroxyl groupof natural nucleosides. Such compounds may be incorporated intoelongating strands by host DNA polymerases, a phenomenon that may beassociated with both carcinogenicity and mitochondrial toxicity.Replacement of hydroxyl groups with fluorine atoms are established inmedicinal chemistry. A well-known example is the replacement of theterminal hydroxyl group of the antibiotic chloramphenicol with afluorine atom, providing the superior drug florfenicol. In the case of61.6 (or 61.8 and 61.9), the fluorine maintains many beneficialH-bonding interactions with RT that the pseudo-3′-hydroxyl of 61.5provided, but will not provide a handle for incorporation into nucleicacids.

Considerations in the chemistry of derivatives 61.8/61.9: A protectedversion of a maybe required (using, e.g., a pivalate) or a maskedversion of A (methoxy in lieu of aniline). If a masked version of a isrequired, synthesis of the base will be required. Yields for theseprocesses in the synthesis of cyclobut-A and G derivatives aregood-to-excellent. Fluorination reactions in the presence of unprotectedA are well precedented to go in good yield (˜90%) when pyridine is usedas a solvent. Acidic deprotection should not cause de-glycosidation ofbase since there is no formal glycosidic linkage (O—C—N).Phosphonylation should proceed as for other derivatives in-house. Allreactions proceed with useful kinetic diastereoselectivity. In baseintroduction reactions, equilibration conditions may be used to improvethe kinetic diastereoselection ratio. All compound made by this routeare racemic. Enantiomerically pure compounds may be prepared by methodsknown to those of skill in the chemical arts.

Illustrated above is a synthetic sequence for the preparation ofcompound 61.16. Other nucleotide bases may optionally be used in thissynthetic sequence.

Example 62 Representative Compounds of Formula 67 Cyclopropyl NucleosideSeries

Representative compounds of the invention can be prepared as illustratedabove. The synthesis of cyclopropyl nucleosides of type 62.18 and 62.19is well documented by Chu and coworkers. Additional syntheses arereported by Csuk et al. Synthetic methods allow for the homochiralproduction of 62.18 and 62.19. Syntheses of compound types 62.17, 62.20,and 62.21 are also reported; these provide for racemic material.

Considerations in the synthesis of 62.17: Literature reports are forindustrial processes. Racemic products: diastereomers are produced by anon-stereoselective cyclopropanation reactions and separation of thedesired isomer with cis cyclopropyl substituents from that with transmay require rigorous separation techniques or alternate syntheticpreparations because of the presence of an additional stereocenter atthe THP anomeric position, as shown above in 62.23.

Considerations in the synthesis of 62.18 and 62.19: For the D series(compound 62.18), synthesis of key intermediate 62.29 (see below) can bepreformed in 10 steps in 6 pots (24% overall yield from an abundantstarting material, 1,2:5,6-di-O-isopropylidine-D-mannitol. Purine basescan be constructed from free amine 62.29. Phosphonate synthesis proceedswell according to known methodology. For the L series, the startingmaterial is vitamin C.

Example 63 Synthesis of Representative Compounds of Formula 68 VinylicNucleoside Series

Representative compounds of the invention can be prepared as illustratedabove. There are only a few reports of compound types 63.33 and 63.34 inthe literature. Most reports provide for the syntheses of trans isomers63.34. The one report that discusses cis isomers 63.33 does not stateclear separation conditions from the mixture of cis and trans compoundsformed. The cis isomers best resembles the geometry of the nucleosideantiviral agents and are therefore important compounds. Modeling studiesindicate that 63.33 will be accommodated by the RT active site. However,when minimized in the RT active site side-by-side with tenofovir, someof the base stacking interactions that provide binding energy betweenthe inhibitor and the template strand may be lost.

Example 64 Synthesis of Representative Compounds of Formula 69

Representative compounds of the invention can be prepared as illustratedabove. In WO 01/10429 A2, the synthesis of 64.1 was fully describedusing D-gulonolactone as the starting material. The secondary alcohol in64.1 can be optionally protected with an appropriate protecting group(e.g. a silyl protecting group) to give 64.2 (See Greene and Wuts,Protective Groups in Organic Synthesis, 1999). Compound 64.3 can beoptionally deprotected to give 64.4 containing the secondary alcohol.Compound 64.4 in an appropriate aprotic solvent can be treated with atleast one equivalent of an appropriate organic or inorganic base. Anappropriate electrophile bearing a leaving group is added to producecompound 64.5. Suitable aprotic solvents include, but are not limitedto, dimethyl formamide, dimethyl sulfoxide, and N-methylpyrrolidinone.Suitable organic or inorganic base include, but are not limited to,sodium hydride, potassium carbonate, and triethylamine. Suitable leavinggroups include, but are not limited to, chlorine, bromine, iodine,p-toluenesulfonate, methanesulfonate, and trifluoromethanesulfonate.

The isopropylidene group in 64.5 can be removed to provide phosphonate64.6. Various procedures for the removal of the isopropylidene group aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,1999. Note that R^(P1) and R^(P2) in 64.5 and 64.6 do not need to be thesame.

A specific preparation of phosphonate 64.6 is illustrated above. Thesecondary alcohol in 64.7 can be protected with t-butyldimethylsilyl(TBDMS) group to give 64.8 (See Greene and Wuts, Protective Groups inOrganic Synthesis, 1999). Compound 64.8 can be alkylated by treating amixture of 64.8 and 64.9 with sodium cyanoborohydride. The aldehyde 64.9can be prepared using the procedure outlined in below.

The tertiary amine 64.10 can be treated with tetrabutylammonium fluoridein THF to remove the TBDMS protecting group. Compound 64.11 can betreated with at least one equivalent of potassium tert-butoxide indimethylformamide. The phosphonate-containing electrophile shown aboveis added to produce 64.12. The isopropylidene group in 64.12 can beremoved by treating 64.12 with an aqueous solution of hydrochloric acidto give 64.13. The para-toluenesulfonate of diethylhydroxymethylphosphonate can be prepared in a single step from diethylhydroxymethylphosphonate and para-toluenesulfonyl chloride in pyridineand diethyl ether (see Holy, et al., Collect. Czech. Chem. Commun. 1982,47, 3447-3463).

Example 65 Synthesis of Representative Compounds of Formulae 70 and 71

Representative compounds of the invention can be prepared as illustratedabove. The synthesis of 65.1 can be performed as outlined in WO 01/10429A2, using D-gulonolactone as the starting material. Treating 65.1 withhydrochloric acid can remove the isopropylidene group. The two secondaryalcohols in diol 65.2 can be non-selectively protected with anappropriate protecting group to generate a mixture of 65.3 and 65.4.These two products can be converted to 65.6 and 65.8, respectively,using procedures described in Example 64 (from 64.1 to 64.6).

The preparation of specific examples of Compounds of Formulae 70 and 71are illustrated above. The details about introducing and removingprotecting groups can be found in Greene and Wuts, Protective Groups inOrganic Synthesis, John Wiley and Sons, Inc., 1999. Other procedures aresimilar to those described in Example 64.

Example 66 Synthesis of Representative Compounds of Formula 72

Representative compounds of the invention can be prepared as illustratedabove. In the above scheme, R⁴ and R⁵ are appropriate protective groups.Group X is either hydroxyl (or oxygen) or thiol (or sulfur), orappropriately protected hydroxyl or thiol. Additionally, R⁵ can be acyclic protecting group for both the hydroxyl and X. The general methodfor the preparation of intermediates 66.3, 66.4, 66.5, and final product66.6, are described in WO 03/020222 A2 page 28 line 10 to page 53 line22, as well as the references cited therein. Additional description isprovided in WO 01/32153 A2 page 41 line 3 to page 56 line 29 andreferences cited therein. Other good sources of information fortransformation from 66.5 to 66.6 are Townsend, Chemistry of Nucleosidesand Nucleotides, Plenum Press, 1994; and Vorbruggen and Ruh-Pohlenz,Handbook of Nucleoside Synthesis, John Wiley & Sons, Inc., 2001.

A specific example for the synthesis of a dioxolane nucleoside analog isillustrated above. Treating the mixture of 66.2.1 and 66.2.2 withp-toluenesulfonic acid, followed by removal of the benzyl protectinggroup on the carboxylic acid produces a mixture of carboxylic acids66.2.3 and 66.2.4. Treating acid 66.2.3 with lead(IV) tetraacetate givesacetate 66.2.5, which can be converted to nucleoside 66.2.6 under thereaction conditional described above. Treating acid 66.2.4 with samereaction procedures for 66.2.3 to 66.2.6 can generate a differentdiastereomer 66.2.8, which is an L-nucleoside analog.

A specific example for the synthesis of a oxathiolane nucleoside analogis illustrated above. The syntheses of 66.3.6 and 66.3.8 are analogousto that of 66.2.6 and 66.2.8 described above.

Preparation and Availability of Starting Materials:

Compound 66.2.1 can be prepared from commercially available startingmaterial 66.4.1 (available from Acros, catalog number 34693-0050 or34693-0250, or from Epsilon, catalog number 95040) following the methodillustrated above. Compound 66.2.2 can be prepared from commerciallyavailable starting material 66.4.2 (Fluka, catalog number 59437)following the method illustrated above. The preparation of 66.3.2 wasdescribed in WO 03/020222 A2 page 34 line 7 to page 36 line 5, andreferences cited therein.

Example 67 Synthesis of Representative Compounds of Formula 73

Representative compounds of the invention can be prepared as illustratedabove. The desired phosphonate substituted analogs are prepared by firstreacting glycal 67.3 (obtained as described in J. Am. Chem. Soc. 1972,94, 3213) with phenylselenyl chloride followed by treatment with therespective phosphonate alcohols 67.4 in the presence of silverperchlorate (J. Org. Chem. 1991, 56, 2642-2647). Oxidation of theresulting chloride using hydrogen peroxide provides the desiredphosphonate 67.2.

For instance, 67.3 dissolved in CH₂Cl₂, is treated with one equivalentof phenyl selenyl chloride at −70° C. followed by silver perchlorate inthe presence of diethyl(hydroxymethyl) phosphonate (67.5). Thephosphonate is transformed into the d4T analog 67.6 by oxidation withhydrogen peroxide. Using the above procedure, but employing differentphosphonate reagents 67.4 in place of 67.5, the corresponding products67.2 bearing different linking groups are obtained. Additionally,analogs containing a variety of bases can be prepared by starting withthe appropriately protected glycals (see examples in: J. Am. Chem. Soc.1972, 94, 3213).

Example 68 Synthesis of Representative Compounds of Formula 74

Representative compounds of the invention can be prepared as illustratedabove. The desired phosphonate substituted analogs are prepared byreaction of d4T (68.1) (obtained as described in U.S. Pat. No. 4,978,655col. 2 ln. 46 to col. 3 ln. 47) with the respective alkylating reagents68.3. Illustrated above is the preparation of phosphonate linkage to d4Tthrough the 5′-hydroxyl group. D4T is dissolved in a solvent such as,but not limited to, DMF or THF, and is treated with a phosphonatereagent bearing a leaving group in the presence of a suitable organic orinorganic base. In compounds 68.3, X is a leaving group such as, but notlimited to, bromide, chloride, iodide, p-toluenesulfonate,trifluoromethanesulfonate, or methanesulfonate.

For instance, 68.1 dissolved in DMF, is treated with one equivalent ofsodium hydride and one equivalent of(toluene-4-sulfonylmethyl)-phosphonic acid diethyl ester 68.4 (preparedaccording to the procedures in J. Org. Chem. 1996, 61, 7697) to give d4Tphosphonate 68.5, in which the linkage is a methylene group. Using theabove procedure, but employing different phosphonate reagents 68.3 inplace of 68.4, the corresponding products 68.2 bearing different linkinggroups are obtained. In a similar manner, using a variety of d4Tspossessing different natural and non-natural nucleoside bases with theappropriate protecting groups, numerous other valuable analogs can beobtained.

Example 69 Synthesis of Representative Compounds of Formula 75

Representative compounds of the invention can be prepared as illustratedin schemes 69.1-69.12 below.

Representative compounds of the invention can be prepared as illustratedabove. The phosphorous-containing compounds of the present invention ofFormula 75 can be readily prepared according to the conventionalsynthetic procedures exemplified in Schemes 69.1-69.12 where aphosphonate-containing moiety may be attached using established chemicalprocesses. The preparations may be carried out from the precursorcompounds described in both WO 02/30930 and WO 02/30931. The methods forthese conversions from precursor compounds to the phosphorous-containingcompounds are described in Comprehensive Organic Transformations,Richard C. Larock, ed, VCH, 1989; Comprehensive Organic Synthesis, BarryM. Trost and Ian Fleming eds., Pergamon Press, 1991. Protection offunction groups during the transformation is described in ProtectiveGroups in Organic Synthesis, Theodora W. Greene and Peter G. M. Wuts,eds., Wiley, 1999.

Example 70 Synthesis of Representative Compounds of Formula 76

Representative compounds of the invention can be prepared as illustratedabove. The core components of this reaction sequence are thetransformations from primary alcohol 70.3 to phosphonate 70.6.Appropriate oxidant(s) can convert the primary alcohol (5′-hydroxy) in70.3 to a carboxylic acid or its corresponding ester. In the case of anester, an additional deprotection step will give the carboxylic acid70.4. A variety of oxidation procedures can be found in the literatureand can be utilized here. These include, but are not limited to, thefollowing methods: (i) pyridinium dichromate in Ac₂O, t-BuOH, anddichloromethane producing the t-butyl ester, followed by deprotectionusing a reagent such as trifluoroacetic acid to convert the ester to thecorresponding carboxylic acid (see Classon, et al., Acta Chem. Scand.Ser. B 1985, 39, 501-504; Cristalli, et al., J. Med. Chem. 1988, 31,1179-1183); (ii) iodobenzene diacetate and2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) inacetonitrile, producing the carboxylic acid (see Epp, et al., J. Org.Chem. 1999, 64, 293-295; Jung et al., J. Org. Chem. 2001, 66,2624-2635); (iii) sodium periodate, ruthenium(III) chloride inchloroform producing the carboxylic acid (see Kim, et al., J. Med. Chem.1994, 37, 4020-4030; Homma, et al., J. Med. Chem. 1992, 35, 2881-2890);(iv) chromium trioxide in acetic acid producing the carboxylic acid (seeOlsson et al.; J. Med. Chem. 1986, 29, 1683-1689; Gallo-Rodriguez etal.; J. Med. Chem. 1994, 37, 636-646); (v) potassium permanganate inaqueous potassium hydroxide producing the carboxylic acid (see Ha, etal., J. Med. Chem. 1986, 29, 1683-1689; Franchetti, et al., J. Med.Chem. 1998, 41, 1708-1715); and (vi) nucleoside oxidase from S.maltophilia to give the carboxylic acid (see Mahmoudian, et al.,Tetrahedron 1998, 54, 8171-8182).

The preparation of 70.5 from 70.4 using lead(IV) tetraacetate (LG=OAc)is described by Teng et al., J. Org. Chem. 1994, 59, 278-280 andSchultz, et al; J. Org. Chem. 1983, 48; 3408-3412. When lead(IV)tetraacetate is used together with lithium chloride (see Kochi, et al.,J. Am. Chem. Soc. 1965, 87, 2052), the corresponding chloride isobtained (70.5, LG=Cl). Lead (IV) tetraacetate in combination withN-chlorosuccinimide can produce the same product (70.5, LG=Cl) (seeWang, et al., Tetrahedron: Asymmetry 1990, 1, 527; and Wilson et al.,Tetrahedron: Asymmetry 1990, 1, 525). Alternatively, the acetate leavinggroup (LG) can also be converted to other leaving group such as bromideby treatment of trimethylsilyl bromide to give 70.5 (see Spencer, etal., J. Org. Chem. 1999, 64, 3987-3995).

The coupling of 70.5 (LG=OAc) with a variety of nucleophiles wasdescribed by Teng et al., Synlett 1996; 346-348 and U.S. Pat. No.6,087,482; column 54 line 64 to column 55 line 20. Specifically, thecoupling between 70.5 and diethyl hydroxymethylphosphonate in thepresence of trimethylsilyl trifluoromethanesulfonate (TMS-OTf) isdescribed. Other compounds with the general structure ofHO-linker-POR^(P1)R^(P2) can also be used so long as the functionalgroups in these compounds are compatible with the coupling reactionconditions. There are many examples in the published literaturedescribing the coupling of 70.5 (LG=halogen) with a variety of alcohols.The reactions can be facilitated with a number of reagents, such assilver(I) salts (see Kim et al.; J. Org. Chem. 1991, 56, 2642-2647;Toikka et al., J. Chem. Soc. Perkins Trans. 1, 1999, 13, 1877-1884),mercury(II) salts (see Veeneman et al.; Recl. Trav. Chim. Pays-Bas.1987, 106, 129-131), boron trifluoride diethyl etherate (see Kunz etal., Hel. Chim Acta 1985, 68, 283-287), tin(II) chloride (see O'Leary etal., J. Org. Chem. 1994, 59, 6629-6636), alkoxide (see Shortnacy-Fowleret al., Nucleosides Nucleotides 2001, 20, 1583-1598), and iodine (seeKartha et al., J. Chem. Soc. Perkins Trans. 1, 2001, 770-772). Thesemethods can be selectively used in conjunction with different methods informing 70.5 with various leaving groups (LG) to produce 70.6.

The introduction and removal of protecting groups is commonly practicedin the art of organic synthesis. Many sources of information ontransformations involving protecting groups are available in thepublished literature, e.g. Greene and Wuts, Protecting Groups in OrganicSynthesis, 3^(rd) Ed., John Wiley & Sons, Inc., 1999. The main purposeis to temporarily transform a functional group so that it will survive aset of subsequent reaction procedures. Afterward, the originalfunctional group can be restored by a preconceived deprotectionprocedure. Therefore, the transformations from 70.1 to 70.2, from 70.2to 70.3, and from 70.6 to 70.7 are intended to allow importanttransformations (e.g., from 70.3 to 70.6) to occur while preserving thefunctional groups that already exist in the core structures.

It should be understood that in the transformation 70.6 to 70.7, R^(P1)and R^(P2) need not remain unchanged. The final form of R^(P1) andR^(P2) can be selected from a variety of possible structures.

The scheme shown above provides a specific example for the generalscheme discussed above. Compound 70.2.1 is prepared using methoddescribed in the patent filing WO 01/90121 (page 115). The 5′-hydroxylin 70.2.1 is protected as a t-butyldimethylsilyl (TBDMS) ether. The 2′-and 3′-hydroxyl groups can be protected as benzoyl (Bz) esters to give70.2.2. The 5′-hydroxyl can then be deprotected to give 70.2.3.Oxidation using iodobenzene diacetate and2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) converts theprimary alcohol to the corresponding acid 70.2.4. Further oxidation of70.2.4 using lead tetraacetate can produce 70.2.5. Coupling between70.2.5 and diethyl hydroxymethylphosphonate (available fromSigma-Aldrich, Cat. No. 39, 262-6) effected by TMS-OTf can afford70.2.6. Treating 70.2.6 with TMS-Br converts the phosphodiester to thecorresponding phosphonic acid 70.2.7. Deprotection of the 2′- and3′-hydroxyl gives 70.2.8 as an example of the generic structure 76,where Base is an adenine, R¹, R⁵, and R⁶ are hydrogen, R² is methylgroup, R³ and R⁴ are hydroxyl groups, linker is a methylene group, andR^(P1) and R^(P2) are both hydroxyl groups.

The phosphonic acids in 70.2.7 and 70.2.8 are illustrative examples.Other forms of phosphonates can be accessed via the phosphonic acid, orother forms, such as the corresponding diesters.

Preparation and Availability of Starting Materials for Examples 70-153

A variety of compounds of the general structure 70.1 can either beprepared using procedures described in the literature, or be purchasedfrom commercial sources. The following are good sources for informationon the art of preparing a variety of compounds of the general structure70.1: Townsend, Chemistry of Nucleosides and Nucleotides, Plenum Press,1994; and Vorbruggen and Ruh-Pohlenz, Handbook of Nucleoside Synthesis,John Wiley & Sons, Inc., 2001.

There are limited number of common precursors that were used to preparethe structures 70.1 in the examples that follow. Many of them aredescribed in the various patents listed at the beginning of thisdocument and the references cited therein. The following is a list ofthese common precursors and their commercial sources or method ofpreparation.

Examples 71-153

Examples 71-153 employ the reaction conditions described above inExample 70. It should be understood that specific reagents, solvents,and reaction conditions used can be substituted by one skilled in theart to accommodate the structure and reactivity requirements of thestarting materials. Alternative methods including, but not limited to,those discussed in Example 70 can be applied as needed. Alternativeprotection and deprotection procedures are also likely to be devised andadapted as needed.

Example 71 Synthesis of Representative Compounds of Formula 76

Example 72 Synthesis of Representative Compounds of Formula 76

Example 73 Synthesis of Representative Compounds of Formula 76

Example 74 Synthesis of Representative Compounds of Formula 76

Example 75 Synthesis of Representative Compounds of Formula 76

Example 76 Synthesis of Representative Compounds of Formula 76

Example 77 Synthesis of Representative Compounds of Formula 76

Example 78 Synthesis of Representative Compounds of Formula 76

Example 79 Synthesis of Representative Compounds of Formula 76

Example 80 Synthesis of Representative Compounds of Formula 76

Example 81 Synthesis of Representative Compounds of Formula 76

Example 82 Synthesis of Representative Compounds of Formula 76

Example 83 Synthesis of Representative Compounds of Formula 76

Example 84 Synthesis of Representative Compounds of Formula 76

Example 85 Synthesis of Representative Compounds of Formula 76

Example 86 Synthesis of Representative Compounds of Formula 76

Example 87 Synthesis of Representative Compounds of Formula 76

Example 88 Synthesis of Representative Compounds of Formula 76

Example 89 Synthesis of Representative Compounds of Formula 76

Example 90 Synthesis of Representative Compounds of Formula 76

Example 91 Synthesis of Representative Compounds of Formula 76

Example 92 Synthesis of Representative Compounds of Formula 76

Example 93 Synthesis of Representative Compounds of Formula 76

Example 94 Synthesis of Representative Compounds of Formula 76

Example 95 Synthesis of Representative Compounds of Formula 76

Example 96 Synthesis of Representative Compounds of Formula 76

Example 97 Synthesis of Representative Compounds of Formula 76

Example 98 Synthesis of Representative Compounds of Formula 76

Example 99 Synthesis of Representative Compounds of Formula 76

Example 100 Synthesis of Representative Compounds of Formula 76

Example 101 Synthesis of Representative Compounds of Formula 76

Example 102 Synthesis of Representative Compounds of Formula 76

Example 103 Synthesis of Representative Compounds of Formula 76

Example 104 Synthesis of Representative Compounds of Formula 76

Example 105 Synthesis of Representative Compounds of Formula 76

Example 106 Synthesis of Representative Compounds of Formula 76

Example 107 Synthesis of Representative Compounds of Formula 76

Example 108 Synthesis of Representative Compounds of Formula 76

Example 109 Synthesis of Representative Compounds of Formula 76

Example 110 Synthesis of Representative Compounds of Formula 76

Example 111 Synthesis of Representative Compounds of Formula 76

Example 112 Synthesis of Representative Compounds of Formula 76

Example 113 Synthesis of Representative Compounds of Formula 76

Example 114 Synthesis of Representative Compounds of Formula 76

Note: one equivalent of TBDMS-Cl can be used in the protection of the5′-hydroxyl. The mixture of two TBDMS ethers of the two primary alcoholscan be separated, and the 5′-hydroxyl protected ether can be used insubsequent reactions.

Example 115 Synthesis of Representative Compounds of Formula 76

Example 116 Synthesis of Representative Compounds of Formula 76

Example 117 Synthesis of Representative Compounds of Formula 76

Example 118 Synthesis of Representative Compounds of Formula 76

Example 119 Synthesis of Representative Compounds of Formula 76

Example 120 Synthesis of Representative Compounds of Formula 76

Example 121 Synthesis of Representative Compounds of Formula 76

Example 122 Synthesis of Representative Compounds of Formula 76

Example 123 Synthesis of Representative Compounds of Formula 76

Example 124 Synthesis of Representative Compounds of Formula 76

Example 125 Synthesis of Representative Compounds of Formula 76

Note: Several options exist for the protection of the amine in thestarting material. It can be protected as its corresponding benzylcarbamate, allyl carbamate, trifluoroacetamide, orN-diphenylmethyleneamine derivative.

Example 126 Synthesis of Representative Compounds of Formula 76

Example 127 Synthesis of Representative Compounds of Formula 76

Example 128 Synthesis of Representative Compounds of Formula 76

Example 129 Synthesis of Representative Compounds of Formula 76

Example 130 Synthesis of Representative Compounds of Formula 76

Example 131 Synthesis of Representative Compounds of Formula 76

Example 132 Synthesis of Representative Compounds of Formula 76

Example 133 Synthesis of Representative Compounds of Formula 76

Example 134 Synthesis of Representative Compounds of Formula 76

Example 135 Synthesis of Representative Compounds of Formula 76

Example 136 Synthesis of Representative Compounds of Formula 76

Example 137 Synthesis of Representative Compounds of Formula 76

Example 138 Synthesis of Representative Compounds of Formula 76

Example 139 Synthesis of Representative Compounds of Formula 76

Example 140 Synthesis of Representative Compounds of Formula 76

Example 141 Synthesis of Representative Compounds of Formula 76

Example 142 Synthesis of Representative Compounds of Formula 76

Example 143 Synthesis of Representative Compounds of Formula 76

Example 144 Synthesis of Representative Compounds of Formula 76

Note: one equivalent of TBDMS-Cl can be used in the protection of the5′-hydroxyl. The mixture of two TBDMS ethers of the two primary alcoholscan be separated, and the 5′-hydroxyl protected ether can be used insubsequent reactions.

Example 145 Synthesis of Representative Compounds of Formula 76

Example 146 Synthesis of Representative Compounds of Formula 76

Note: one equivalent of TBDMS-Cl can be used in the protection of the5′-hydroxyl. The mixture of two TBDMS ethers of the two primary alcoholscan be separated, and the 5′-hydroxyl protected ether can be used insubsequent reactions.

Example 147 Synthesis of Representative Compounds of Formula 76

Example 148 Synthesis of Representative Compounds of Formula 76

Example 149 Synthesis of Representative Compounds of Formula 76

Example 150 Synthesis of Representative Compounds of Formula 76

Example 151 Synthesis of Representative Compounds of Formula 76

Example 152 Synthesis of Representative Compounds of Formula 76

Example 153 Synthesis of Representative Compounds of Formula 76L-Nucleoside Analogs

Many compounds of Formula 76 with a sugar moiety in its L-configurationare either commercially available or can be prepared by proceduresdescribed in the published literature. Many of the compounds illustratedin Examples 71-153 can be prepared from one of the precursors describedin Example 70 (see section: Preparation And Availability Of StartingMaterials). The enantiomers of other nucleoside analogs (theL-nucleosides) can be prepared from enantiomers of the precursors of70.3.1, 70.3.2, and 70.3.3. This Example describes a preparation of theenantiomers of 70.3.1, 70.3.2, and 70.3.3.

The commercially available starting material 153.4.1 can be converted to153.4.4, which is the enantiomer of 70.3.1, using the sequence ofreactions outlined above. The osmium tetroxide catalyzed dihydroxylationintroduces the diol selectively to the face opposite of thetert-butyldimethylsilyl (TBDMS) ether of the hydroxymethyl group.

The diol in intermediate 153.4.3 can be protected as its TBDMS ether.Diisobutylaluminum hydride reduction of the lactone at low temperatureproduces 153.4.5, which can be converted to 153.4.6 by acetylation.

Deprotection of 153.4.6 produces L-ribose (153.4.7). Acylation convertsall hydroxyl groups in 153.4.7 to the corresponding benzoyl esters.Standard coupling reactions with a variety of nucleobases produces153.4.10, which is the enantiomer of 70.3.3.

From 70.3.1, 70.3.2, and 70.3.3, L-nucleosides can be prepared usingknown procedures, many of which are discussed in previous sections, inthe patents cited, and in the published literature.

Many compounds in Examples 70-153 have their corresponding L-analogstarting materials described in the same patent. These L-nucleosides canthen be used in the same reaction sequences to produce the phosphonateanalogs of the L-nucleosides.

Example 154 Synthesis of Representative Compounds of Formulae 84 and 85

Representative compounds of the invention can be prepared as illustratedabove. Direct alkylation of entecavir derivative 154.5 with aphosphonate attached to a leaving group can be performed in the presenceof a suitable organic or inorganic base to obtain analogs of the types154.2 and 154.3. Compound 154.5 is prepared from protected ordeprotected intermediates described in U.S. Pat. No. 5,206,244 and U.S.Pat. No. 5,340,816. After reaction, a mixture of compounds 154.2 and154.3 is furnished, which are separated by the appropriatechromatographic method.

For instance, entecavir (154.1) is treated with sodium hydroxide andreacted with diethyl phosphomethyltriflate to afford a mixture of 154.6and 154.7 as illustrated above. Silica gel chromatography is employed togive pure samples of the separated products.

Example 155 Synthesis of Representative Compounds of Formulae 84 and 85

Representative compounds of the invention can be prepared as illustratedabove. Compounds having the structure 155.4 are prepared fromintermediate 155.8, which is derived from deprotected intermediatesdescribed in U.S. Pat. No. 5,206,244 and U.S. Pat. No. 5,340,816. Diol155.8 is converted to glycal 155.9 through published procedures. Upontreatment with [Br in the presence of the appropriate phosphonatealcohol, glycal 155.9 is converted to iodide 155.10. Nystedmethylenation provides alkene 155.12, whose hydroxy stereocenter is theninverted to give the final compound 155.4.

For instance, intermediate 155.13 is converted to glycal 155.14 (see J.Am. Chem. Soc. 1972, 94, 3213) and then treated with IBr and diethylphosphomethanol to furnish iodide 155.15 (see J. Org. Chem. 1991, 56,2642). Nucleophilic substitution of the iodide using AgOAc affordsacetate 155.16. After methylenation using the procedure of Nysted (U.S.Pat. No. 3,865,848; Aldrichim. Acta 1993, 26, 14), the acetate group isremoved using sodium methoxide in methanol. The resulting alcohol isinverted by the Mitsunobo protocol, and a second acetate deprotectionproduces the desired compound 155.18.

Example 156 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid diisopropyl ester

A mixture of7-hydroxy-6-(4-hydroxy-3-methyl-but-2-enyl)-5-methoxy-4-methyl-3H-isobenzofuran-1-one156a (50 mg, 0.18 mmol, Pankiewicz et al., J. Med. Chem., 45, 703),diisopropyl bromomethylphosphonate (93 mg, 0.36 mmol) and lithiumt-butoxide (1M in THF, 0.54 mL) in DMF (3 mL) was heated at 70° C. for 5hours. The reaction was quenched with 1N HCl. The mixture was pouredinto 5% aqueous lithium chloride, extracted with ethyl acetate, andconcentrated. The residue was purified by chromatography on silica gel,affording[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid diisopropyl ester 156B (25 mg, 32%); ¹H NMR (300 MHz, CDCl₃)

1.25 (m, 12H), 1.79 (s, 3H), 2.05 (s, 3H), 3.37 (d, J=6.6 Hz, 2H), 3.58(d, 2H), 3.77 (s, 3H), 3.97 (m, 2H), 4.68 (m, 2H), 5.19 (s, 2H), 5.45(t, J=6.6 Hz, 1H), 7.83 (s, 1H) ppm.

[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid and[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicAcid Monoisopropyl Ester

To a solution of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid diisopropyl ester 156B (25 mg, 0.055 mmol) and 2,6-lutidine (0.18mL, 1.65 mmol) in acetonitrile was added trimethylsilyl bromide (0.126mL, 1.1 mmol) at 0° C. The mixture was allowed to warm to roomtemperature and stirred for 4 hours. The reaction was quenched withmethanol at 0° C., and the resulting mixture was concentrated. Theresidue was purified by preparative reverse-phase HPLC to afford, afterremoval of the solvent,[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid 156C as an oil (17 mg, 83%); ¹H NMR (300 MHz, CD₃OD) δ 1.81 (s,3H), 2.06 (s, 3H), 3.40 (d, J=6.6 Hz, 2H), 3.50 (d, 2H), 3.77 (s, 3H),3.97 (s, 2H), 5.20 (s, 2H), 5.47 (t, J=6.6 Hz, 1H) and[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid monoisopropyl ester 156D as an oil (2 mg, 7%); ¹H NMR (300 MHz,CD₃OD)

1.23 (d, 6H), 1.81 (s, 3H), 2.08 (s, 3H), 3.40 (d, J=6.6 Hz, 2H), 3.50(d, 2H), 3.77 (s, 3H), 3.90 (s, 2H), 4.50 (m, 1H), 5.20 (s, 2H), 5.47(t, J=6.6 Hz, 1H) ppm.

Example 157 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[5-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-penta-1,3-dienyl]-phosphonicAcid Dimethyl Ester

To a solution of tetramethylmethylene diphosphonate (102 mg, 0.44 mmol)in THF (2.5 mL) was added a THF solution of sodiumbis(trimethysilyl)amide (1.0 M, 0.44 mL). After stirring for 30 minutes,a solution of4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enal157A (30 mg, 0.11 mmol, Pankiewicz et al., J. Med. Chem., 45, 703) inTHF (2.5 mL) was added and stirring was continued for an additional 15minutes. The reaction was quenched with saturated aqueous ammoniumchloride. The mixture was extracted with ethyl acetate. Afterevaporation of solvent, the residue was purified by chromatography onsilica gel eluting with ethyl acetate (50% to 100%)/hexanes, affording[5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-penta-1,3-dienyl]-phosphonicacid dimethyl ester 157B (30 mg, 71%) as an oil; ¹H NMR (300 MHz, CDCl₃)δ 1.80 (s, 3H), 2.04 (s, 3H), 3.45 (d, J=6.6 Hz, 2H), 3.76 (s, 3H), 3.88(d, 6H), 5.20 (s, 3H), 5.55 (m, 1H), 5.95 (m, 1H), 7.05 (m, 1H), 7.65(s, 1H) ppm.

[5-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-penta-1,3-dienyl]-phosphonicAcid

To a solution of[5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-penta-1,3-dienyl]-phosphonicacid dimethyl ester 157B (22 mg, 0.057 mmol) and 2,6-lutidine (0.22 mL,1.71 mmol) in acetonitrile was added trimethylsilyl bromide (0.183 mL,1.71 mmol) at 0° C. The mixture was allowed to warm to room temperatureand stirred for 1 hour. The reaction was quenched with methanol at 0°C., and the resulting mixture was concentrated. The residue was purifiedby preparative reverse-phase HPLC to afford, after removal of thesolvent,[5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-penta-1,3-dienyl]-phosphonicacid 157C as a solid (13 mg, 65%); ¹H NMR (300 MHz, CD₃OD) δ 1.91 (s,3H), 2.10 (s, 3H), 3.55 (d, J=6.6 Hz, 2H), 3.75 (s, 3H), 5.2 (s, 2H),5.6-5.8 (m, 2H), 6.9 (m, 1H) ppm.

Example 158 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

6-(4-Bromo-3-methyl-but-2-enyl)-7-hydroxy-5-methoxy-4-methyl-3H-isobenzofuran-1-one

Polymer-supported triphenylphosphine (3 mmol/g, 0.5 g) was soaked indichloromethane (10 mL) for 1 hour.7-Hydroxy-6-(4-hydroxy-3-methyl-but-2-enyl)-5-methoxy-4-methyl-3H-isobenzofuran-1-one158A (100 mg, 0.36 mmol) and carbon tetrabromide (143 mg, 0.43 mmol)were sequentially added and the mixture was shaken for 1 h at roomtemperature. More carbon tetrabromide (143 mg, 0.43 mmol) was added andthe mixture was shaken further for 1 hour. The mixture was filtered andthe filtrate was concentrated. The residue was chromatographed on silicagel (0% to 60% ethyl acetate/hexanes) to afford6-(4-bromo-3-methyl-but-2-enyl)-7-hydroxy-1-methoxy-4-methyl-3H-isobenzofuran-1-one158B as an oil (52 mg, 42%); ¹H NMR (300 MHz, CDCl₃) δ 1.95 (s, 3H),2.16 (s, 3H), 3.44 (d, J=7.2 Hz, 2H), 3.78 (s, 3H), 3.98 (s, 2H), 5.21(s, 2H), 5.68 (t, J=7.2 Hz, 1H), 7.71 (brs, 1H) ppm.

[5-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicAcid Diethyl Ester

n-Butyl lithium (1.6 M in hexanes, 1 mL) was added to an equal volume ofTHF at −20° C. A solution of diethyl methylphosphonate (220 mg, 1.45mmol) in THF (1 mL) was then added dropwise and the solution was stirredfor 30 minutes. After cooling at −60° C., the solution was transferredvia a cannula to a vial containing copper (I) iodide (276 mg, 1.45mmol), and the resulting mixture was stirred for 1 h at −30° C. Asolution of6-(4-bromo-3-methyl-but-2-enyl)-7-hydroxy-5-methoxy-4-methyl-3H-isobenzofuran-1-one158B (50 mg, 0.15 mmol) in THF (1 mL) was added and the mixture wasallowed to warm to 0° C. for 2 h before saturated aqueous ammoniumchloride was added. The reaction mixture was acidified with 2 N HCl andextracted with ethyl acetate. The ethyl acetate extract was concentratedand the residue was chromatographed on silica gel (40% to 100% ethylacetate/hexanes), affording[5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicacid diethyl ester 158C as an oil (27 mg, contaminated with the startingdiethyl methylphosphonate); ¹H NMR (300 MHz, CDCl₃) δ 1.32 (m, 6H),1.8-1.9 (m, 5H), 2.18 (s, 3H), 2.25 (m, 2H), 3.42 (d, J=7.2 Hz, 2H),3.78 (s, 3H), 4.15 (m, 4H), 5.21 (s, 2H), 5.24 (t, J=7.2 Hz, 1H), 7.65(s, 1H) ppm.

[5-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicAcid Monoethyl Ester

A mixture of[5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicacid diethyl ester 158C (27 mg, 9.066 mmol), LiOH (200 mg), MeOH (3 mL)and water (1 mL) was stirred at 70° C. for 4 hours. After cooling, thereaction solution was acidified with 2 N HCl, mixed with brine, andextracted with ethyl acetate/acetonitrile. The organic extract wasconcentrated and the residue was purified by preparative reverse-phaseHPLC (acetonitrile and 0.1% aqueous CF₃COOH), affording[5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicacid monoethyl ester 158D (7 mg, 28%); ¹H NMR (300 MHz, CD₃OD) δ 1.28(t, J=6.9 Hz, 3H), 1.7-1.9 (m, 5H), 2.20 (s, 3H), 2.2-2.3 (m, 2H), 3.41(d, J=6.6 Hz, 2H), 3.80 (s, 3H), 4.02 (m, 2H), 5.2-5.3 (m, 3H) ppm.

Compound 158E can be prepared as illustrated below.

[5-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicAcid

To a solution of{5-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-3-methyl-pent-3-enyl}-phosphonicacid diethyl ester (20 mg, 0.039 mmol) in DMF (0.5 mL) and DCM (0.5 mL)was added TMSBr (50.5 μL, 0.39 mmol) followed by 2,6-lutidine (45.3 μL,0.39 mmol). The reaction was allowed to proceed for one hour when it wascomplete, as judged by LCMS. The reaction mixture was quenched with MeOHand concentrated to dryness. The residue was purified by preparativereverse-phase HPLC. The fraction containing the desired product wasconcentrated and treated with 10% TFA/DCM for 5 minutes. Afterconcentration, the residue was purified by preparative reverse-phaseHPLC to provide 7 mg (50%) of[5-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicacid as a solid. ¹H NMR (300 MHz, CD₃OD) δ 1.66-1.78 (m, 5H), 2.10 (s,3H), 2.16-2.22 (m, 2H), 3.34 (d, J=7.2 Hz, 2H), 3.72 (s, 3H), 5.16 (s,2H), 5.20 (t, J=7.2 Hz, 1H) ppm; ³¹P (121.4 MHz, CD₃OD) δ 31.57 ppm; MS(m/z) 355 [M−H]⁻, 357 [M+H]⁺.

Example 159 Preparation of Representative Compounds of the Invention

Representative compounds of the invention can be prepared as illustratedbelow.

2-(4-Bromo-but-2-enyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid Methyl Ester

To a cooled (−78° C.) solution of mycophenolic acid methyl ester 159A(138 mg, 0.41 mmol) in THF (2.5 mL) was added a THF solution of sodiumbis(trimethysilyl)amide (1.0 M, 0.98 mL). After stirring for 30 minutes,a solution of 1,4-dibromo-2-butene (950 mg, 4.1 mmol) in THF (2.5 mL)was added and stirring was continued for 10 minutes. The resultingmixture was warmed to −30° C. and stored at this temperature for 16hours. The reaction was quenched with saturated aqueous ammoniumchloride. The mixture was extracted with ethyl acetate to give, afterevaporation of the solvent, a residue which was purified bychromatography on silica gel eluting with ethyl acetate (0% to40%)/hexanes, affording2-(4-bromo-but-2-enyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester 159B (150 mg, 78%) as an oil; ¹H NMR (300 MHz, CDCl₃)δ 1.75 (s, 3H), 2.0-2.4 (m, 8H), 2.62 (m, 1H), 3.37 (d, J=6.6 Hz, 2H),3.58 (s, 3H), 3.76 (s, 3H), 3.88 (d, J=4.8 Hz, 2H), 5.1-5.3 (m, 3H),5.67 (brs, 2H), 7.67 (s, 1H) ppm.

2-[4-(Diethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid Methyl Ester

A solution of2-(4-bromo-but-2-enyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester 159B (140 mg, 0.30 mmol) and triethylphosphite (600mg, 3.6 mmol) in toluene (30 mL) was stirred at reflux for 20 hours. Themixture was concentrated and chromatographed on silica gel eluting withethyl acetate (60% to 100%)/hexanes, affording2-[4-(diethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester 159C as an oil (70 mg, 43%); ¹H NMR (300 MHz, CDCl₃)

1.27 (m, 6H), 1.79 (s, 3H), 2.0-2.7 (m, 8H), 3.37 (d, J=6.6 Hz), 3.52(s, 3H), 3.75 (s, 3H), 4.08 (m, 4H), 5.20 m, 3H), 5.45 (m, 2H) ppm.

2-[4-(Diethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

A mixture of2-[4-(diethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester 159C (33 mg, 0.063 mmol) and lithium hydroxide (44 mg)in a mixture of THF (6 mL) and water (1 mL) was stirred at roomtemperature for 6 hours. The organic solvent was removed and the residuewas partitioned between ethyl acetate and 5% aqueous sodium bicarbonate.The aqueous layer was acidified with 2 N HCl and extracted with ethylacetate. The ethyl acetate extract was concentrated, affording2-[4-(diethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid 159D as an oil (30 mg, 100%); ¹H NMR (300 MHz, CDCl₃) δ 1.27 (m,6H), 1.79 (s, 3H), 2.0-2.7 (m, 8H), 3.37 (d, J=6.6 Hz), 3.75 (s, 3H),4.08 (m, 4H), 5.19 (s, 2H), 5.25 (m, 1H), 5.44 (m, 1H), 5.55 (m, 1H),5.45 (m, 2H) ppm.

2-[4-(Ethoxy-hydroxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

A mixture of2-[4-(diethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester 159C (25 mg, 0.048 mmol) and lithium hydroxide (200mg) in a mixture of methanol (3 mL) and water (1 mL) was stirred at 70°C. for 2 hours. The organic solvent was evaporated and the residueacidified with 2N HCl and extracted with ethyl acetate/acetonitrile. Theorganic extract was concentrated and the residue was purified bypreparative reverse-phase HPLC (acetonitrile and 0.1% aqueous CF₃COOH),affording2-[4-(ethoxy-hydroxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid 159E as an oil (15 mg, 89%); ¹H NMR (300 MHz, CD₃OD) δ 1.25 (t,J=6.9 Hz, 3H), 1.81 (s, 3H), 2.1-2.6 (m, 8H), 3.40 (d, J=6.6 Hz, 2H),3.77 (s, 3H), 3.97 (m, 2H), 5.1-5.3 (m, 3H), 5.67 (brs, 2H) ppm.

2-[4-(Dimethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid Methyl Ester

Under a N₂ atmosphere, a solution of2-(4-bromo-but-2-enyl)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester (490 mg, 1.05 mmol) in trimethylphosphite (2.5 mL,21.1 mmol) was heated at 120° C. for 1 hour. The reaction was allowed tocool to room temperature. The reaction mixture was worked up by removalof the solvent in vacuo followed by chromatography using EtOAc-hexanesto provide 460 mg (88%) of the product as an oil. ¹H NMR (300 MHz,CDCl₃) δ 1.77 (s, 3H), 2.081-2.31 (m, 4H), 2.15 (s, 3H), 2.52 (d, 1H,J=22 Hz), 2.54 (d, 1H, J=22 Hz), 2.55-2.63 (m, 1H), 3.36 (d, 2H, J=7Hz), 3.57 (s, 3H), 3.72 (d, 6H, J=11 Hz), 3.76 (s, 3H), 5.20 (s, 2H),5.20-5.26 (m, 1H), 5.36-5.56 (m, 2H), 7.69 (s, 1H) ppm; ³¹P (121.4 MHz,CDCl₃) δ 30.1 ppm; MS (m/z) 497.2 [M+H]⁺, 519.2 [M+Na]⁺.

Compound 159F can be prepared as illustrated below.

2-[4-(Dimethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

2-[4-(Dimethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester (460 mg, 0.927 mmol) in a solution of 1:1:2 of H₂O,MeOH, THF (8 mL) was stirred with LiOH.H₂O (78 mg, 1.86 mmol) at ambienttemperature for 12 hours. A second batch of LiOH.H₂O (40 mg, 0.952 mmol)was added. The reaction mixture was stirred at room temperature foranother 16 hours, after which no further progress was observed. Thereaction was quenched by addition of a saturated aqueous solution ofNH₄Cl. The organic layer was removed in vacuo and the product wasextracted with EtOAc from the aqueous layer, which had been acidified byaddition of 5 drops of 2 N HCl. The product was further purified bychromatography to provide the desired product. ¹H NMR (300 MHz, CDCl₃) δ1.79 (s, 3H), 2.08-2.38 (m, 4H), 2.15 (s, 3H), 2.53 (d, 1H, J=22 Hz),2.60 (d, 1H, J=22 Hz), 2.57-2.64 (m, 1H), 3.38 (d, 2H, J=7 Hz), 3.72 (d,6H, J=11 Hz) 3.76 (s, 3H), 5.20 (s, 2H), 5.27 (t, 1H, J=6 Hz), 5.36-5.63(m, 2H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 30.5 ppm; MS (m/z) 481.2 [M−H]⁻.

Compound 159G can be prepared as illustrated below.

2-[4-(2-[4-(Dihydroxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

To a solution of2-[4-(dimethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid (25 mg, 0.052 mmol) in acetonitrile (2 mL) was added 2,6-lutidine(60 μL, 0.52 mmol) and TMSBr (67 μL, 0.52 mmol). The reaction wasallowed to proceed for 45 minutes when it was completed as judged byLCMS. The reaction mixture was concentrated under reduced pressure andquenched with an aqueous NaOH solution (1 mL). The product was purifiedby RP HPLC (using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA) to provide 14.2 mg (60%) of the product as asolid. ¹H NMR (300 MHz, CD₃OD) δ 1.81 (s, 3H), 2.081-2.31 (m, 4H), 2.16(s, 3H), 2.45 (d, 1H, J=22 Hz), 2.47 (d, 1H, J=22 Hz), 2.55-2.63 (m,1H), 3.38 (d, 2H, J=7 Hz), 3.77 (s, 3H), 5.25 (s, 2H), 5.20-5.36 (m,1H), 5.36-5.56 (m, 2H) ppm; ³¹P (121.4 MHz, CD₃OD) δ 25.4 ppm; MS (m/z)453 [M−H]⁻.

2-[4-(Dimethoxy-phosphoryl)-but-2-enyl]-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl Ester

A solution of2-[4-(dimethoxy-phosphoryl)-but-2-enyl]-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid (160 mg, 0.332 mmol) and trimethylsilylethanol (160 mg, 1.36 mmol)in THF (8.00 mL) was stirred with triphenylphosphine (345 mg, 1.33mmol). To this solution was added diethyl azodicarboxylate (230 μL, 1.33mmol) at 0° C. The mixture was allowed to warm to room temperature andstirred for 16 hours. Additional triphenylphosphine (180 mg, 0.692mmol), trimethylsilylethanol (160 mg, 1.36 mmol), and diethylazodicarboxylate (115 μL, 0.665 mmol) were added and the reactionmixture was stirred for another 1 day at room temperature. The reactionwas worked up by removing the solvents in vacuo and purifying theresidue by silica gel chromatography to provide 192 mg (85%) of theproduct as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s, 9H), 0.05 (s,9H), 0.93-0.96 (m, 2H), 1.20-1.29 (m, 2H), 1.78 (s, 3H), 2.01-2.32 (m,4H), 2.17 (s, 3H), 2.51 (d, 1H, J=22 Hz), 2.58 (d, 1H, J=22 Hz),2.50-2.60 (m, 1H), 3.37 (d, 2H, J=7 Hz), 3.72 (d, 6H, J=11 Hz), 3.76 (s,3H), 4.08 (appt t, 2H, J=8 Hz), 4.30 (appt t, 2H, J=8 Hz), 5.12 (s, 2H),5.15-5.25 (m, 1H), 5.36-5.63 (m, 2H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 29.3ppm; MS (m/z) 705.3 [M+Na]⁺.

2-[4-(Hydroxy-methoxy-phosphoryl)-but-2-enyl]-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl Ester

A mixture of2-[4-(dimethoxy-phosphoryl)-but-2-enyl]-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl ester (184 mg, 0.270 mmol) intert-butylamine (2.8 mL, 27 mmol) was heated at 60° C. for 24 hours. Thesolution was allowed to cool to room temperature and concentrated. Theresidue was purified by silica gel column chromatography usingMeOH/CH₂Cl₂ (0-30%) to provide 75 mg of the product as a clear oil. ¹HNMR (300 MHz, CDCl₃) δ 0.01 (s, 9H), 0.04 (s, 9H), 0.89 (appt t, 2H, J=9Hz), 1.23 (appt t, 2H, J=9 Hz), 1.77 (s, 3H), 2.01-2.31 (m, 4H), 2.17(s, 3H), 2.36 (d, 1H, J=22 Hz), 2.38 (d, 1H, J=22 Hz), 2.52 (septet, 1H,J=9 Hz), 3.39 (d, 2H, J=7 Hz), 3.51 (d, 3H, J=11 Hz), 4.01-4.08 (m, 2H),4.30 (dd, 2H, J=8, 9 Hz), 5.11 (s, 2H), 5.19 (br t, 1H, J=6 Hz),5.33-5.56 (m, 2H), 8.49 (br s, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 22.1ppm; MS (m/z) 667.4 [M+Na]⁺.

2-{4-[(1-Ethoxycarbonyl-ethoxy)-methoxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl Ester

A solution of2-[4-(hydroxy-methoxy-phosphoryl)-but-2-enyl]-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl ester (67 mg, 0.10 mmol) and PyBOP (234mg, 0.450 mmol) in DMF (1.5 mL) was stirred with ethyl (S)-(−)-lactate(53 mg, 0.45 mmol) and DIEA (174 μL, 1.00 mmol) at ambient temperaturefor 1 hour, when complete consumption of the starting materials wasobserved. The reaction was worked up by addition of saturated aqueoussodium chloride and ethyl acetate. The organic layer was separated andwashed with 5% aqueous solution of lithium chloride. The organic layerwas dried in vacuo and the residue was purified by silica gelchromatography using MeOH—CH₂Cl₂ (0-20%) to provide 57 mg (74%) of thedesired product as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ 0.02 (s, 9H),0.05 (s, 9H), 0.88-0.94 (m, 2H), 1.20-1.30 (m, 2H), 1.29 (t, 3H, J=7Hz), 1.45 (d, 3H, J=7 Hz), 1.78 (s, 3H), 2.01-2.31 (m, 4H), 2.17 (s,3H), 2.50-2.58 (m, 1H), 2.65 (d, 1H, J=22 Hz), 2.67 (d, 1H, J=22 Hz),3.39 (d, 2H, J=7 Hz), 3.69 and 3.77 (d, 3H, J=11 Hz), 3.76 (s, 3H), 4.07(appt t, 2H, J=7 Hz), 4.20 (dq, 2H, J=3, 7 Hz), 4.29 (appt t, 2H, J=9Hz), 4.85-4.99 (m, 1H), 5.12 (s, 2H), 5.19 (br t, 1H, J=6 Hz), 5.33-5.61(m, 2H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 28.9, 29.9 ppm; MS (m/z) 791.4[M+Na]⁺.

Compound 159H can be prepared as illustrated below.

2-{4-[(1-Ethoxycarbonyl-ethoxy)-methoxy-phosphoryl]-but-2-enyl}-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

A solution of2-{4-[(1-ethoxycarbonyl-ethoxy)-methoxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl ester (14 mg, 0.018 mmol) in THF (1 mL)was stirred with a 1M solution of TBAF in THF (55 μL, 0.055 mmol) for 1hour. The reaction mixture was concentrated, acidified with 1N HCl andextracted with EtOAc. The organic layer was washed with brine and dried.The product was purified by silica gel column chromatography EtOH-EtOAc(0-10%). Further purification was performed by dissolving the product inCH₂Cl₂ and passing the compound through a 13 mm Acrodisc syringe filterwith a 0.45 μm Nylon membrane to provide 8 mg (77%) of the product. ¹HNMR (300 MHz, CDCl₃) δ 0.92 (t, 3H, J=7 Hz), 1.30 (d, 3H, J=8 Hz), 1.79(s, 3H), 2.10-2.39 (m, 4H), 2.15 (s, 3H), 2.53 (d, 1H, J=8 Hz), 2.65 (d,1H, J=22 Hz), 2.68 (d, 1H, J=22 Hz), 3.38 (d, 2H, J=7 Hz), 3.70 and 3.74(d, 3H, J=11 Hz), 3.76 (s, 3H), 4.07 (m, 2H), 4.96 (dq, 1H, J=7 Hz),5.20 (s, 2H), 5.27 (br t, 1H, J=7 Hz), 5.33-5.55 (m, 2H), 7.51-7.56 (m,1H), 7.68-7.74 (m, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 29.0, 30.1 ppm; MS(m/z) 569.2 [M+H]⁺, 591.3 [M+Na]⁺.

2-{4-[(1-Carboxy-ethoxy)-hydroxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicAcid 2-trimethylsilanyl-ethyl Ester

A solution of2-{4-[(1-ethoxycarbonyl-ethoxy)-methoxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanylethyl ester (12 mg, 0.016 mmol) intert-butylamine (1 mL, 9.6 mmol) was heated at 65° C. for 16 hours. Thesolution was allowed to cool to room temperature and concentrated toprovide the crude product as an oil. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s,9H), 0.04 (s, 9H), 0.86-0.98 (m, 2H), 1.22-1.33 (m, 2H), 1.50 (d, 3H,J=7 Hz), 1.78 (s, 3H), 2.05-2.30 (m, 4H), 2.10 (s, 3H), 2.48-2.63 (m,3H), 3.40 (d, 2H, J=7 Hz), 3.76 (s, 3H), 4.08 (appt t, 2H, J=9 Hz),4.25-4.33 (m, 2H), 4.75-4.84 (m, 1H), 5.13 (s, 2H), 5.15-5.23 (m, 1H),5.33-5.55 (m, 2H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 28.9 ppm; MS (m/z) 725.3[M−H]⁻.

Compound 159I can be prepared as illustrated below.

2-{4-[(1-Carboxy-ethoxy)-hydroxy-phosphoryl]-but-2-enyl}-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

A solution of crude2-{4-[(1-carboxy-ethoxy)-hydroxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl ester (AC-2101-59) and tetrabutylammoniumfluoride in THF (1M, 54 μL, 0.054 mmol) was stirred with THF (1 mL) for2 hours at ambient temperature, when more tetrabutylammonium fluoride inTHF (54 μL, 0.054 mmol) was added. The reaction was stirred for anadditional 16 hours, by which time the reaction was complete. Thereaction mixture was concentrated in vacuo and the product was purifiedby RP HPLC using a Phenomenex Synergi 5μ Hydro RP 80A column (50×21.2mm) with eluents of H₂O, 0.1% TFA-CH₃CN, 0.1% TFA to provide the product(8.0 mg) as a clear oil. ¹H NMR (300 MHz, CDCl₃) δ 1.51 (d, 3H, J=7 Hz),1.79 (s, 3H), 2.05-2.40 (m, 4H), 2.11 (s, 3H), 2.49-2.71 (m, 3H), 3.38(d, 2H, J=6 Hz), 3.76 (s, 3H), 4.85 (br s, 1H), 5.20 (s, 2H), 5.21-5.30(m, 1H), 5.33-5.63 (m, 2H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 27.7 ppm; MS(m/z) 525.2 [M−H]⁻.

Compound 159J can be prepared as illustrated below.

2-{4-[(1-Ethoxycarbonyl-ethylamine)-methoxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl Ester

A solution of2-[4-(hydroxy-methoxy-phosphoryl)-but-2-enyl]-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl ester (20 mg, 0.030 mmol), PyBOP (62.4 mg,0.120 mmol) in DMF (1.0 mL) was stirred with L-alanine ethyl esterhydrochloride (18 mg, 0.12 mmol) and DIEA (26 μL, 0.15 mmol) at ambienttemperature for 1 hour, when complete consumption of the startingmaterials was observed. The reaction was worked up by addition of wateruntil the reaction solution became cloudy. DMF was added dropwise untilthe mixture became clear again. The reaction mixture was filteredthrough Acrodisc (13 mm syringe filter with a 0.45 μm Nylon membrane)and purified by RP HPLC using a Phenomenex Synergi 5μ Hydro RP 80Acolumn (50×21.2 mm), eluting with water and acetonitrile. The fractionscontaining the product were pooled together and concentrated in vacuo toremove the acetonitrile. The remaining solution was saturated withsodium chloride and extracted with EtOAc and acetonitrile to provide 7.2mg of the product. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s, 9H), 0.05 (s, 9H),0.923 (appt t, 2H, J=8 Hz), 1.18-1.31 (m, 5H), 1.41 (t, 3H, J=7 Hz),1.78 (s, 3H), 2.03-2.36 (m, 4H), 2.18 (s, 3H), 2.43-2.63 (m, 3H),3.10-3.30 (m, 1H), 3.40 (d, 2H, J=7 Hz), 3.62 and 3.65 (d, 3H, J=11 Hz),3.76 (s, 3H), 4.03-4.12 (m, 2H), 4.20 (dq, 2H, J=2, 7 Hz), 4.29 (appt t,2H, J=8 Hz), 5.12 (s, 2H), 5.18-5.28 (m, 1H), 5.33-5.67 (m, 2H) ppm; ³¹P(121.4 MHz, CDCl₃) δ 30.4, 31.2 ppm; MS (m/z) 790.4 [M+Na]⁺.

2-{4-[(1-Ethoxycarbonyl-ethylamine)-methoxy-phosphoryl-but-2-enyl}-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicAcid

To a solution of2-{4-[(1-ethoxycarbonyl-ethylamine)-methoxy-phosphoryl]-but-2-enyl}-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanyl-ethyl ester (7.2 mg, 9.38 mmol) in THF (1 mL)was added TBAF (40 μL, 1M solution in THF) at room temperature. Thereaction mixture was stirred for 20 minutes, when the starting materialwas completely converted to the desired product as judged by LCMS. Thereaction mixture was dried in vacuo and re-dissolved in DMF. The productwas purified by RP HPLC using a Phenomenex Synergi 5μ Hydro RP 80Acolumn (50×21.2 mm) with eluents of H₂O—CH₃CN. The fractions containingthe desired product were pooled and further purified on Dowex 50WX8-400packed on a 4.5 cm×2 cm column to elute the sodium salt at H₂O—MeOH(1:1), providing 3.2 mg of the desired product. ¹H NMR (300 MHz, CD₃OD)δ 1.26 (dd, 3H, J=4, 7 Hz), 1.37 (t, 3H, J=8 Hz), 1.80 (s, 3H),2.00-2.22 (m, 4H), 2.10 (s, 3H), 2.25-2.60 (m, 3H), 3.37 (d, 2H, J=7Hz), 3.60 and 3.65 (d, 3H, J=11 Hz), 3.74 (s, 3H), 3.83-3.96 (m, 1H),4.18 (q, 2H, J=8 Hz), 5.15 (s, 2H), 5.25-5.42 (m, 2H), 5.55-5.69 (m, 1H)ppm; ³¹P (121.4 MHz, CD₃OD) δ 33.8, 34.2 ppm; MS (m/z) 568.2 [M+H]⁺,590.3 [M+Na]⁺.

Compound 159K can be prepared as illustrated below.

6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-[4-(hydroxy-methoxy-phosphoryl)-but-2-enyl]-4-methyl-hex-4-enoicAcid

To a solution of2-[4-(hydroxy-methoxy-phosphoryl)-but-2-enyl]-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid 2-trimethylsilanylethyl ester (11 mg, 0.016 mmol) in THF (1 mL) wasadded TBAF (50 μL, 1M solution in THF) at room temperature. The solutionwas stirred for 16 hours and concentrated. The solution was dried underreduced pressure and re-suspended in DMF (0.8 mL) and water (0.25 mL).The solution was filtered through Acrodisc (13 mm syringe filter with a0.45 μm Nylon membrane) and purified by RP HPLC using a PhenomenexSynergi 5μ Hydro RP 80A column (50×21.2 mm) with eluents of H₂O, 0.1%TFA-CH₃CN, 0.1% TFA. The product from the column was subjected to ionexchange chromatography (Sodium salt form of Dowex 50WX8-400) using a2×4.5 cm column eluting with H₂O-MeOH (1:1) to provide 7.5 mg of thedesired product as an oil. ¹H NMR (300 MHz, CDCl₃) δ 1.80 (s, 3H),2.01-2.29 (m, 5H), 2.11 (s, 3H), 2.35 (d, 2H, J=22 Hz), 3.38 (d, 2H, J=7Hz), 3.53 (d, 3H, J=11 Hz), 3.75 (s, 3H), 5.19 (s, 2H), 5.26 (t, 1H, J=6Hz), 5.43-5.54 (m, 2H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 23.5 ppm; MS (m/z)469.2 [M+H]⁺, 491.3 [M+Na]⁺.

Example 160 Preparation of Representative Compounds of the Invention

Representative compounds of the invention can be prepared as illustratedbelow.

6-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicAcid Methyl Ester

To a solution of6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoicacid methyl ester (222 mg, 0.66 mmol), triphenylphosphine (260 mg, 0.996mmol), and diethyl azodicarboxylate (173 mg, 0.996 mmol) in THF (3 mL)at 0° C. was added a solution of 2-trimethylsilylethanol (142 μL, 0.996mmol) in THF (3 mL). The resulting yellow solution was allowed to warmto room temperature and stirred overnight. The reaction was concentratedto dryness and ether and hexanes were added. Triphenylphosphine oxidewas removed by filtration and the filtrate was concentrated and purifiedby silica gel chromatography to provide 248 mg of the desired product asa colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s, 9H), 1.18-1.30 (m,2H), 1.81 (s, 3H), 2.18 (s, 3H), 2.25-2.33 (m, 2H), 2.37-2.45 (m, 2H),3.42 (d, 2H, J=7 Hz), 3.62 (s, 3H), 3.77 (s, 3H), 4.25-4.35 (m, 2H),5.13 (s, 2H), 5.12-5.22 (m, 1H) ppm.

[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde

A solution of6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid methyl ester (618 mg, 1.42 mmol) in MeOH (10 mL), CH₂Cl₂ (10 mL)and pyridine (50 μL, 0.618 mmol) was cooled to −70° C. using a dryice/acetone bath according to the procedure of Smith, D. B. et al., J.Org. Chem., 1996, 61, 6, 2236. A stream of ozone was bubbled through thereaction via a gas dispersion tube until the reaction became blue incolor (15 minutes). The ozone line was replaced with a stream ofnitrogen and bubbling continued for another 15 minutes, by which timethe blue color had disappeared. To this solution at −70° C. was addedthiourea (75.7 mg, 0.994 mmol) in one portion, and the cooling bath wasremoved. The reaction was allowed to warm to room temperature andstirred for 15 hours. The reaction was worked up by filtration to removesolid thiourea S-dioxide, and then partitioned between CH₂Cl₂ and water.The organic layer was removed. The aqueous layer was washed with CH₂Cl₂one more time and the organic extracts were combined. The organic layerwas washed with aqueous 1N HCl, saturated NaHCO₃ and brine. The organicextracts were dried in vacuo and the residue was purified to by silicagel chromatography to afford 357 mg (75%) of the product as a whitesolid. ¹H NMR (300 MHz, CDCl₃) δ −0.01 (s, 9H), 1.05-1.15 (m, 2H), 2.15(s, 3H), 3.69 (s, 3H), 3.78 (d, 2H, J=1 Hz), 4.27-4.39 (m, 2H), 5.11 (s,2H), 9.72 (d, 1H, J=1 Hz) ppm.

4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal

[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde(70 mg, 0.21 mmol) in toluene (2 mL) was heated at 100° C. with2-(triphenyl-phosphanylidene)-propionaldehyde (72.9 mg, 0.23 mmol)overnight. A second portion of2-(triphenyl-phosphanylidene)-propionaldehyde (33 mg, 0.11 mmol) wasadded and the reaction mixture was heated for an additional day. Afterconcentration, the residue was purified by silica gel chromatography toprovide 54 mg (83%) of the desired product as a pale yellow oil. ¹H NMR(300 MHz, CDCl₃) δ 0.00 (s, 9H), 1.10-1.21 (m, 2H), 1.87 (s, 3H), 2.16(s, 3H), 3.67-3.76 (m, 2H), 3.74 (s, 3H), 4.27-4.39 (m, 2H), 5.11 (s,2H), 6.40-6.48 (m, 1H), 9.2 (s, 1H) ppm.

6-(4-Hydroxy-3-methyl-but-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

A solution of4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(103 mg, 0.27 mmol) in methanol (5 mL) was cooled to 0° C. A solution ofCeCl₃ (0.68 mL, MeOH: H₂O, 9:1) was added, followed by LiBH₄ (0.14 mL,0.28 mmol of a 2M solution in THF). The ice bath was removed and thereaction mixture was allowed to warm to room temperature. The reactionmixture was stirred for an additional 40 minutes whereupon TLC indicatedcomplete consumption of starting aldehyde. The reaction was worked up byaddition of aqueous 1N HCl (0.5 mL) and the product was extracted withCH₂Cl₂. The organic layer was washed with saturated aqueous sodiumbicarbonate solution and brine. The organic layer was concentrated underreduced pressure and the residue was purified by silica gelchromatography to provide 100 mg (97%) of the product as a clear liquid.¹H NMR (300 MHz, CDCl₃) δ 0.00 (s, 9H), 1.20 (dd, 2H, J=7, 8 Hz), 1.81(s, 3H), 2.13 (s, 3H), 3.38-3.50 (m, 2H), 3.74 (s, 3H), 3.95 (s, 2H),4.27 (dd, 2H, J=7, 8 Hz), 5.08 (s, 2H), 5.17-5.44 (m, 1H) ppm.

6-(2-Hydroxy-ethyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

To a solution of[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde(97 mg, 0.29 mmol) in THF (5 mL) was added an aliquot of a 2 M LiBH₄ inTHF (150 μL, 0.300 mmol). The reaction mixture was stirred at roomtemperature for 1 hour when complete consumption of the startingmaterials was observed by TLC. The reaction mixture was worked up byaddition of an aqueous 1N HCl solution and extraction with EtOAc. Theorganic layer was dried in vacuo and the residue was purified by silicagel chromatography to provide the product. ¹H NMR (300 MHz, CDCl₃) δ0.00 (s, 9H), 1.20 (dd, 2H, J=7, 9 Hz), 2.07 (br s, 1H), 2.14 (s, 3H),2.97 (t, 2H, J=6 Hz), 3.76 (t, 2H, J=6 Hz), 3.77 (s, 3H), 4.32 (dd, 2H,J=7, 8 Hz), 5.08 (s, 2H) ppm.

{2-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilany-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-ethoxymethyl}-phosphonicAcid Diisopropyl Ester

A mixture of6-(2-hydroxy-ethyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(79 mg, 0.23 mmol) was heated with bromomethylphosphonic aciddiisopropyl ester (120 mg, 0.46 mmol) in the presence of lithiumt-butoxide (22 mg, 0.27 mmol) in DMF (2 mL) at 70° C. overnight. Thereaction mixture was purified by RP HPLC (acetonitrile and 0.1% aqueousCF₃COOH) to provide the desired product. ¹H NMR (300 MHz, CDCl₃) δ 0.00(s, 9H), 1.13-1.25 (m, 2H), 1.26 (t, 12H, J=6 Hz), 2.12 (s, 3H), 2.98(t, 2H, J=7 Hz), 3.60-3.73 (m, 4H), 3.77 (s, 3H), 4.05-4.16 (m, 2H),4.62-4.74 (m, 2H), 5.07 (s, 2H) ppm; MS (m/z) 539 [M+Na]⁺.

[2-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-ethoxymethyl]-phosphonicAcid

To a solution of{2-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-ethoxymethyl}-phosphonicacid diisopropyl ester (7.5 mg, 0.014 mmol) in acetonitrile (2 mL) and2,6-lutidine (25 μL, 0.21 mmol) was added trimethylsilyl bromide (27 μL,0.21 mmol) at room temperature. The reaction was allowed to proceed for18 hours when completion of the reaction was indicated by LCMS. Thereaction was quenched by addition of MeOH and concentration. The residuewas purified by RP-HPLC using a C18 column. The collected product wasdissolved in a solution of 10% TFA/CH₂Cl₂ to assure completedeprotection. The reaction mixture was lyophilized to provide thedesired product. ¹H NMR (300 MHz, CD₃OD) δ 2.12 (s, 3H), 2.98 (t, 2H,J=7 Hz), 3.66-3.76 (m, 4H), 3.78 (s, 3H), 5.21 (s, 2H) ppm; MS (m/z) 331[M−H]⁻.

Example 161 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

6-(4-Bromo-3-methyl-but-2-enyl)-7-hydroxy-5-methoxy-4-methyl-3H-isobenzofuran-1-one

Polymer-supported triphenylphosphine (3 mmol/g, 0.5 g) was soaked indichloromethane (10 mL) for 1 hour.7-Hydroxy-6-(4-hydroxy-3-methyl-but-2-enyl)-5-methoxy-4-methyl-3H-isobenzofuran-1-one(100 mg, 0.36 mmol) and carbon tetrabromide (143 mg, 0.43 mmol) wereadded sequentially and the mixture was shaken for 1 hour at roomtemperature. More carbon tetrabromide (143 mg, 0.43 mmol) was added andthe mixture was shaken further for 1 hour. The mixture was filtered andthe filtrate was concentrated. The residue was chromatographed on silicagel (0% to 60% ethyl acetate/hexanes) to afford6-(4-bromo-3-methyl-but-2-enyl)-7-hydroxy-5-methoxy-4-methyl-3H-isobenzofuran-1-oneas an oil (52 mg, 42%); ¹H NMR (300 MHz, CDCl₃) δ 1.95 (s, 3H), 2.16 (s,3H), 3.44 (d, J=7.2, 2H), 3.78 (s, 3H), 3.98 (s, 2H), 5.21 (s, 2H), 5.68(t, J=7.2 Hz, 1H), 7.71 (brs, 1H) ppm.

[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phosphonicAcid Dimethyl Ester

A solution of6-(4-bromo-3-methyl-but-2-enyl)-7-hydroxy-5-methoxy-4-methyl-3H-isobenzofuran-1-one(33 mg, 0.097 mmol) in trimethylphosphite (1.0 mL, 8.5 mmol) was heatedto 100° C. for 1 hour, whereupon complete reaction was indicated byLCMS. The reaction was worked up by removal of the excess reagent underreduced pressure and the residue was purified by silica gelchromatography using EtOAc-hexanes (20-100%) to provide 20 mg (60%) ofthe desired product. ¹H NMR (300 MHz, CDCl₃) δ 1.90 (s, 3H), 2.09 (s,3H), 2.48 (d, 2H, J=22 Hz), 3.38 (t, 2H, J=6 Hz), 3.64 (d, 6H, J=11 Hz),3.72 (s, 3H), 5.14 (s, 2H), 5.33 (q, 1H, J=6 Hz), 7.65 (br s, 1H) ppm;MS (m/z) 371 [M+H]⁺.

Example 162 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phosphonicAcid

To a solution of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phosphonicacid dimethyl ester (18 mg, 0.049 mmol) in acetonitrile (2 mL) was addedTMSBr (63 μL, 0.49 mmol) and 2,6-lutidine (85 μL, 0.73 mmol) at 0° C.The reaction solution was allowed to warm to room temperature andstirred for 2 hours when completion of the reaction was observed byLCMS. The reaction was cooled to 0° C. and quenched by the addition ofMeOH. The reaction mixture was concentrated under reduced pressure andthe residue was purified by RP HPLC using a C18 column with a gradientof H₂O-acetonitrile (5-0%) over 20 minutes to provide 12.2 mg (73%) ofthe product. ¹H NMR (300 MHz, CD₃OD) δ 1.95 (s, 3H), 2.15 (s, 3H), 2.48(d, 2H, J=22 Hz), 3.44 (t, 2H, J=6 Hz), 3.79 (s, 3H), 5.24 (s, 2H), 5.38(q, 1H, J=7 Hz), 6.87 (br s, 1H) ppm; MS (m/z) 341 [M−H]⁻.

Example 163 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid monophenyl ester and[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicAcid Diphenyl Ester

To a solution of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid (49 mg, 0.13 mmol) in DMF (0.4 mL) and phenol (62 mg, 0.65 mmol)was added dicyclohexyl carbodiimide (107 mg, 0.52 mmol) and DMAP (8 mg,0.065 mmol) in DMF (0.6 mL), slowly at 0° C. The reaction was allowed towarm to room temperature and heated to 140° C. for 10 hours. Aftercooling to room temperature the mixture was filtered and extracted withaqueous 1N NaOH solution. The aqueous layer was acidified with aqueous1N HCl and extracted with EtOAc. The organic layer was dried over Na₂SO₄and concentrated to dryness. The residue was purified by RP HPLC toprovide 18.5 mg of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid monophenyl ester (major product) as a pale yellow solid and 4.1 mgof[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid diphenyl ester (minor product) also as a pale yellow solid. Majorproduct: ¹H NMR (300 MHz, CD₃OD) δ 1.82 (s, 3H), 2.16 (s, 3H), 3.46 (d,2H, J=7 Hz), 3.70 (d, 2H, J=8 Hz), 3.77 (s, 3H), 3.96 (s, 2H), 5.25 (s,2H), 5.52 (t, 1H, J=8 Hz), 7.10-7.21 (m, 3H), 7.30 (t, 2H, J=8 Hz) ppm;³¹P (121.4 MHz, CD₃OD) δ 17.3 ppm; MS (m/z) 449.0 [M+H]⁺, 471.2 [M+Na]⁺.Minor product: ¹H NMR (300 MHz, CD₃OD) δ 1.82 (s, 3H), 2.15 (s, 3H),3.47 (d, 2H, J=7 Hz), 3.77 (s, 3H), 3.98-4.06 (m, 4H), 5.25 (s, 2H),5.50-5.61 (m, 1H), 7.10-7.25 (m, 6H), 7.30-7.41 (m, 4H) ppm; ³¹P (121.4MHz, CD₃OD) δ 16.3 ppm; MS (m/z) 525.2 [M+H]⁺, 547.2 [M+Na]⁺.

Example 164 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

2-{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phenoxy-phosphinoyloxy}-propionicacid ethyl ester

To a solution of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid monophenyl ester (18.5 mg, 0.040 mmol) and ethyl (S)-(−)-lactate(47 μL, 0.400 mmol) in pyridine (0.5 mL) was added PyBOP (32 mg, 0.060mmol). The solution was stirred at room temperature for 1 hour, when anadditional portion of PyBOP (21 mg, 0.040 mmol) was added. The solutionwas stirred for another hour and concentrated. The residue was purifiedby HPLC to provide 7.5 mg of the desired product as a clear oil. ¹H NMR(300 MHz, CD₃OD) δ 1.22 and 1.25 (t, 3H, J=7 Hz), 1.42 and 1.50 (d, 3H,J=7 Hz), 1.82 and 1.83 (s, 3H), 2.16 (s, 3H), 3.47 (d, 2H, J=7 Hz), 3.78(s, 3H), 3.89 (d, 1H, J=8 Hz), 3.93-4.02 (m, 3H), 4.10-4.22 (m, 2H),4.94-5.08 (m, 1H), 5.25 (s, 2H), 5.50-5.60 (m, 1H), 7.15-7.27 (m, 3H),7.33-7.41 (m, 2H) ppm; ³¹P (121.4 MHz, CD₃OD) δ 18.9, 20.3 ppm(diastereomers at phosphorus); MS (m/z) 549.2 [M+H]⁺, 571.3 [M+Na]⁺.

Example 165 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

2-{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phenoxy-phosphinoylamino}-propionicAcid Ethyl Ester

To a solution of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid monophenyl ester (20 mg, 0.045 mmol) and L-alanine ethyl esterhydrochloride (68.5 mg, 0.45 mmol) in pyridine (1.0 mL) was added PyBOP(70 mg, 0.14 mmol). After stirring overnight, the mixture wasconcentrated and the residue purified by RP HPLC using a C18 column witha gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 3.6 mg ofthe product as a colorless gel. ¹H NMR (300 MHz, CD₃OD) δ 1.17-1.3 (m,6H), 1.8-1.9 (m, 3H), 2.16 (s, 3H), 3.17 (m, 1H), 3.47 (d, 2H), 3.72-3.8(m, 5H), 3.92-4.2 (m, 4H), 5.25 (s, 2H), 5.54 (m, 1H), 7.18 (m, 3H),7.33 (m, 2H) ppm; ³¹P (121.4 MHz, CD₃OD) δ 24.1, 25.0 ppm (diastereomersat phosphorus); MS (m/z) 546.2 [M−H]⁺.

Example 166 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid monomethyl ester

To a solution of[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid diphenyl ester (53 mg, 0.1 mmol) in methanol (0.5 mL) was added anaqueous solution of 1N NaOH (300 μL). After stirring overnight, themixture was concentrated and the residue purified by RP HPLC using a C18column with a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA toprovide 5 mg of the product as a colorless gel, together with thephosphonic acid monophenyl ester (7 mg) and the phosphonic acid dimethylester (14.5 mg). ¹H NMR (300 MHz, CD₃OD) δ 1.84 (s, 3H), 2.16 (s, 3H),3.47 (d, 2H, J=7 Hz), 3.6 (d, 2H, J=12 Hz), 3.75 (d, 3H, J=11 Hz), 3.79(s, 3H), 3.94 (s, 2H), 5.26 (s, 2H), 5.53 (t, 1H, J=7 Hz) ppm; ³¹P(121.4 MHz, CD₃OD) δ 21.5 ppm; MS (m/z) 385.2 [M−H]⁺, 387.1 [M+H]⁺.

Example 167 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

(2-{4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicAcid Diethyl Ester

To a solution of4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(84 mg, 0.22 mmol), (2-amino-ethyl)-phosphonic acid diethyl esteroxalate (91 mg, 0.33 mmol), and sodium triacetoxyborohydride (93 mg,0.44 mmol) in DMF (1.5 mL) was added acetic acid (60 μL, 1.0 mmol) atroom temperature. The solution was stirred for 2 days when it wasquenched by addition of saturated aqueous sodium bicarbonate solutionand EtOAc. The organic layer was separated and concentrated underreduced pressure. The residue was purified by RP HPLC using a C18 columnwith a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 115mg (96%) of the product as an oil. ¹H NMR (300 MHz, CDCl₃) δ 0.04 (s,9H), 1.16-1.27 (m, 2H), 1.34 (t, 6H, J=7 Hz), 1.94 (s, 3H), 2.18 (s,3H), 2.20-2.31 (m, 2H), 3.13-3.31 (m, 2H), 3.48 (d, 2H, J=7 Hz), 3.54(s, 2H), 3.78 (s, 3H), 4.14 (pent, 4H, J=7 Hz), 4.30-4.37 (m, 2H), 5.13(s, 2H), 5.65 (t, 1H, J=7 Hz), 6.23 (br s, 2H) ppm; ³¹P (121.4 MHz,CDCl₃) δ 27.8 ppm; MS (m/z) 542.3 [M+H]⁺, 564.2 [M+Na]⁺.

{2-[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-ethyl}-phosphonicAcid

A solution of(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (30 mg, 0.055 mmol), TMSBr (72 μL, 0.55 mmol), and2,6-lutidine (64 μL, 0.55 mmol) was stirred in CH₂Cl₂ (1 mL) and DMF(0.5 mL) for 1 hour at ambient temperature. The reaction mixture waspurified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 7.8 mg of the product as a whitesolid. ¹H NMR (300 MHz, CD₃OD) δ 1.96 (s, 3H), 1.95-2.07 (m, 2H), 2.16(s, 3H), 3.10-3.24 (m, 2H), 3.51 (d, 2H, J=7 Hz), 3.57 (s, 2H), 3.81 (s,3H), 5.25 (s, 2H), 5.73 (t, 1H, J=7 Hz) ppm; ³¹P (121.4 MHz, CD₃OD) δ20.2 ppm; ¹⁹F NMR (282.6 MHz, CD₃OD) δ −74.0 ppm; MS (m/z) 386.3 [M+H]⁺.

Example 168 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[2-(Methanesulfonyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicAcid Diethyl Ester

A solution of(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (45 mg, 0.092 mmol) in CH₂Cl₂ (0.5 mL) was stirredwith methanesulfonyl chloride (21 μL, 0.28 mmol) and pyridine (45 μL,0.55 mmol) at ambient temperature overnight. The reaction was quenchedby addition of 2 drops of water. The reaction mixture was concentratedand purified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 36 mg of the product (63%) as aclear gel. ¹H NMR (300 MHz, CDCl₃) δ 0.05 (s, 9H), 1.18-1.29 (m, 2H),1.29 (t, 6H, J=7 Hz), 1.85 (s, 3H), 2.00-2.13 (m, 2H), 2.19 (s, 3H),2.85 (s, 3H), 3.32-3.43 (m, 2H), 3.47 (d, 2H, J=7 Hz), 3.69 (s, 2H),3.79 (s, 3H), 4.05 (pent, 4H, J=7 Hz), 4.30-4.37 (m, 2H), 5.13 (s, 2H),5.45 (t, 1H, J=7 Hz) ppm; ³¹P (121.4 MHz, CD₃Cl) δ 27.5 ppm; MS (m/z)642.2 [M+Na]⁺.

(2-{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-methanesulfonyl-amino}-ethyl)-phosphonicAcid

A solution of[2-(methanesulfonyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicacid diethyl ester (18 mg, 0.029 mmol) in acetonitrile (0.5 mL) wasstirred with TMSBr (38 μL, 0.29 mmol) and 2,6-lutidine (34 μL, 0.29mmol) for 2 hours at room temperature. The reaction was worked up byaddition of EtOAc and aqueous 1N HCl. The organic layer was washed withbrine and the solvent was removed in vacuo. The residue was suspended ina solution of 10% TFA-CH₂Cl₂ for 10 minutes before it was dried toprovide 9.9 mg of the desired product (73%) as a white solid. ¹H NMR(300 MHz, DMSO-d6) δ 1.76 (s, 3H), 1.76-1.88 (m, 2H), 2.10 (s, 3H), 2.87(s, 3H), 3.24-3.35 (m, 2H), 3.39 (d, 2H, J=7 Hz), 3.65 (s, 2H), 3.75 (s,3H), 5.22 (s, 2H), 5.41-5.48 (m, 1H) ppm; ³¹P (121.4 MHz, DMSO-d6) δ21.4 ppm; MS (m/z) 464.1 [M+H]⁺.

Example 169 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[2-(Acetyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicAcid Diethyl Ester

To a solution of(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (32 mg, 0.059 mmol) in acetic acid (0.5 mL) was addedacetic anhydride (0.5 mL). The solution was stirred at room temperaturefor 90 minutes when it was quenched by addition of 2 drops of water. Thesolution was dried in vacuo and the residue was purified by RP HPLCusing a C18 column with a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1%TFA to provide 28 mg of the product (81%) as a clear gel. The NMR dataof this compound shows two rotamers in a ratio of 70:30. ¹H NMR (300MHz, CDCl₃) δ 0.05 (s, 9H), 1.17-1.27 (m, 2H), 1.30 and 1.31 (t, 6H, J=7Hz), 1.70-1.79 (m, 2H), 1.76 (s, 3H), 2.00 (s, 3H), 2.18 (s, 3H),3.40-3.52 (m, 2H), 3.46 (d, 2H, J=7 Hz), 3.77 (s, 3H), 3.79 and 3.93 (s,3H), 4.07 (pent, 4H, J=7 Hz), 4.27-4.35 (m, 2H), 5.13 (s, 2H), 5.22-5.30(m, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 27.5 and 28.9 ppm; MS (m/z) 584.1[M+H]⁺, 606.2 [M+Na]⁺.

(2-{Acetyl-[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-amino}-ethyl)-phosphonicAcid

To a solution of[2-(acetyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicacid diethyl ester (14 mg, 0.024 mmol) in acetonitrile (0.5 mL) wasadded TMSBr (31 μL, 0.24 mmol) and 2,6-lutidine (28 μL, 0.24 μmmol). Thesolution was stirred at room temperature for 1 hour. The reaction wasquenched by addition of methanol and aqueous 1N HCl. The product wasextracted with EtOAc. The combined organic extracts were dried overNa₂SO₄ and concentrated in vacuo. The product was purified by RP HPLCusing a C18 column with a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1%TFA to provide 5.4 mg of the product (53%) as a white solid. The NMRdata of this compound shows two rotamers. ¹H NMR (300 MHz, CDCl₃) δ 1.67and 1.73 (s, 3H), 1.85-2.12 (m, 5H), 2.13 (s, 3H), 3.30-3.61 (m, 4H),3.75 (s, 3H), 3.76 (br s, 2H), 5.17 (s, 2H), 5.31 (br s, 1H) ppm; ³¹P(121.4 MHz, CDCl₃) δ 27.5 and 28.8 ppm; MS (m/z) 428.2 [M+H]⁺, 450.2[M+Na]⁺.

Example 170 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[2-(Benzyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicAcid Diethyl Ester

A solution of(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (30 mg, 0.055 mmol), benzaldehyde (5.6 μL, 0.055mmol), and sodium triacetoxyborohydride (23 mg, 0.11 mmol) was stirredwith acetic acid (15.7 μL, 0.28 mmol) in DMF (0.5 mL) at roomtemperature over night. The reaction was quenched with a 10% aqueousNa₂CO₃ solution and the product was extracted with EtOAc. The organiclayer was dried and concentrated under reduced pressure. The product waspurified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 15 mg of the product (43%) as aclear gel. ¹H NMR (300 MHz, CDCl₃) δ 0.02 (s, 9H), 1.18-1.25 (m, 2H),1.24 (t, 6H, J=7 Hz), 1.86 (s, 3H), 1.88-2.02 (m, 2H), 2.16 (s, 3H),2.65-2.74 (m, 2H), 3.93 (s, 2H), 3.46 (br d, 4H, J=7 Hz), 3.76 (s, 3H),4.00 (pent, 4H, J=7 Hz), 4.25-4.34 (m, 2H), 5.11 (s, 2H), 5.34-5.43 (m,1H), 7.18-7.33 (m, 5H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 30.9 ppm; MS (m/z)632.4 [M+H]⁺, 654.3 [M+Na]⁺.

(2-{Benzyl-[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-amino}-ethyl)-phosphonicAcid

A solution of(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-3-ethyl)-phosphonicacid diethyl ester (15 mg, 0.024 mmol) in acetonitrile (0.5 mL) wastreated with TMSBr (31 μL, 0.24 mmol) and 2,6-lutidine (28 μL, 0.24mmol). The solution was stirred at ambient temperature for 1 hour, whenit was quenched with methanol. The solvent was removed under reducedpressure and the residue was purified by RP HPLC using a C18 column witha gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 11 mg ofthe product (93%) as a white solid. ¹H NMR (300 MHz, CD₃OD) δ 1.89 (s,3H), 2.03-2.15 (m, 2H), 2.14 (s, 3H), 3.30-3.47 (m, 2H), 3.50 (br s,2H), 3.62 (br s, 2H), 3.79 (s, 3H), 4.28 (s, 2H), 5.23 (s, 2H), 5.76 (brs, 1H), 7.46 (br s, 5H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 20.1 ppm; MS (m/z)476.3 [M+H]⁺, 498.3 [M+Na]⁺.

Example 171 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[2-(Formyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicAcid Diethyl Ester

To a solution of(2-{4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (74 mg, 0.14 mmol) in formic acid (1 mL) was addedformic anhydride (1 mL) and the solution was stirred at room temperaturefor 1 hour. The reaction mixture was concentrated and the crude productcarried onto the next step. The NMR data of this compound shows tworotamers with the ratio of 70:30. ¹H NMR (300 MHz, CDCl₃) δ 0.05 (s,9H), 1.18-1.28 (m, 2H), 1.28 and 1.30 (t, 6H, J=7 Hz), 1.74 (s, 3H),1.84-2.08 (m, 2H), 2.19 (s, 3H), 3.34-3.45 (m, 2H), 3.47 (d, 2H, J=7Hz), 3.72 and 3.87 (s, 2H), 3.78 and 3.79 (s, 3H), 4.06 and 4.07 (pent,4H, J=7 Hz), 4.26-4.37 (m, 2H), 5.13 (s, 2H), 5.30-5.46 (m, 1H), 8.03and 8.19 (s, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 27.5 and 28.1 ppm; MS(m/z) 570.1 [M+H]⁺, 592.2 [M+Na]⁺.

(2-{Formyl-[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-amino}-ethyl)-phosphonicAcid

To a solution of crude[2-(formyl-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-amino)-ethyl]-phosphonicacid diethyl ester (78 mg, 0.14 mmol) in acetonitrile (1 mL) was addedTMSBr (177 μL, 1.4 mmol) and 2,6-lutidine (163 μL, 1.4 mmol). Thesolution was stirred at room temperature for 1 hour when it was quenchedby addition of methanol and 1N aqueous HCl. The product was extractedwith EtOAc and purified by RP HPLC using a C18 column with a gradient ofH₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 29 mg of the product asa white solid. The NMR data of this compound shows two rotamers with theratio of approximately 70:30. ¹NMR (300 MHz, CD₃OD) δ 1.62 and 1.64 (s,3H), 1.83-1.98 (m, 2H), 2.16 (s, 3H), 3.38-3.55 (m, 4H), 3.78 (s, 3H),3.80 and 3.91 (s, 2H), 5.22 (s, 2H), 5.39-5.52 (m, 1H), 8.03 and 8.18(s, 1H) ppm; MS (m/z) 414.2 [M+H]⁺, 436.2 [M+Na]⁺.

Example 172 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

({4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-methyl)-phosphonicAcid Diethyl Ester

To a solution of4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(500 mg, 1.33 mmol), (2-aminomethyl)phosphonic acid diethyl esteroxalate (376 mg, 1.46 mmol), sodium triacetoxyborohydride (563 mg, 2.66mmol) in DMF (10 mL) was added acetic acid (380 μL, 6.65 mmol) at roomtemperature. The solution was stirred overnight when it was quenched byaddition of saturated aqueous sodium bicarbonate solution and EtOAc. Theorganic layer was separated and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography to provide 500 mg(71%) of the product as an oil. ¹H NMR (300 MHz, CDCl₃) δ 0.00 (s, 9H),1.13-1.23 (m, 2H), 1.25 and 1.27 (t, 6H, J=7 Hz), 1.65-1.75 (m, 2H),1.77 (s, 3H), 2.13 (s, 3H), 2.80 (s, 1H), 3.14 (s, 2H), 3.41 (d, 2H, J=7Hz), 3.73 (s, 3H), 4.08 and 4.09 (pent, 4H, J=7 Hz), 4.20-4.30 (m, 2H),5.08 (s, 2H), 5.30 (t, 1H, J=7 Hz) ppm; ³¹P (121.4 MHz, CDCl₃) δ 26.5ppm; MS (m/z) 528.1 [M+H]⁺, 550.2 [M+Na]⁺.

{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-methyl}-phosphonicAcid

To a solution of({4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-methyl)-phosphonicacid diethyl ester (20 mg, 0.038 mmol) in DMF (0.5 mL) was added TMSBr(49 μL, 0.38 mmol) and 2,6-lutidine (44 μL, 0.38 mmol). The solution wasstirred at room temperature for 1 hour when it was quenched by additionof methanol. The product was purified by RP HPLC using a C18 column witha gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 5.6 mg ofthe product as a white solid. ¹H NMR (300 MHz, CD₃OD and CDCl₃) δ 1.93(s, 3H), 2.13 (s, 3H), 2.94 (br d, 2H, J=11 Hz), 3.42-3.53 (m, 2H), 3.60(s, 2H), 3.78 (s, 3H), 5.22 (s, 2H), 5.71 (br s, 1H) ppm; ³¹P (121.4MHz, CDCl₃) δ 8.5 ppm; MS (m/z) 372.2 [M+H]⁺, 743.2 [2M+H]⁺.

Example 173 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

2-({2-[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-ethyl}-phenoxy-phosphinoyloxy)-propionicAcid Ethyl Ester

A solution of4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(188 mg, 0.5 mmol) was stirred with2-[(2-aminoethyl)phenoxy-phosphinoyloxy]-propionic acid ethyl esteracetic acid salt (315.8 mg, 0.75 mmol) in CH₂Cl₂ (3 mL) for 2 hours atambient temperature. Sodium triacetoxyborohydride (159 mg, 0.75 mmol)was added to the solution and the reaction was allowed to proceed for 1hour. The reaction was quenched by addition of a saturated aqueoussolution of NaHCO₃ and the product was extracted with EtOAc. The organiclayer was removed under reduced pressure and the residue was resuspendedin a 10% TFA/CH₂Cl₂ for 1 hour. The reaction mixture was concentratedand the product was purified by RP HPLC using a C18 column with agradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 198 mg ofthe product as a white solid. The NMR data of this compound shows twodiastereomers at phosphorus in a ratio of approximately 45:55. ¹H NMR(300 MHz, CD₃OD) δ 1.23 and 1.24 (t, 3H, J=7 Hz), 1.38 and 1.52 (d, 3H,J=7 Hz), 1.97 and 1.98 (s, 3H), 2.14 (s, 3H), 2.44-2.66 (m, 2H),3.31-3.48 (m, 2H), 3.51 (d, 2H, J=7 Hz), 3.66 (d, 2H, J=5 Hz), 3.80 (s,3H), 4.10-4.27 (m, 2H), 4.90-5.10 (m, 1H), 5.20 (s, 2H), 5.73-5.82 (m,1H), 7.15-7.27 (m, 3H), 7.35-7.45 (m, 2H) ppm; ³¹P (121.4 MHz, CD₃OD) δ22.6, 24.3 ppm; MS (m/z) 561.9 [M+H]⁺.

Example 174 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

2-[Hydroxy-(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphinoyloxy]-propionicAcid Ethyl Ester

A solution of4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(38 mg, 0.1 mmol) was stirred with2-[(2-aminoethyl)-phenoxy-phosphinoyloxy]-propionic acid ethyl esteracetic acid (63 mg, 0.15 mmol) in CH₂Cl₂ (1 mL) for 2 hours at ambienttemperature. Sodium triacetoxyborohydride (32 mg, 0.15 mmol) was addedto the solution and the reaction was allowed to proceed for 1 hour. Thereaction was quenched by addition of a saturated aqueous solution ofNaHCO₃ and the product was extracted with EtOAc. The organic layer wasremoved under reduced pressure and the residue was re-suspended in 10%TFA/CH₂Cl₂ for 1 hour. The reaction mixture was concentrated and theproduct was purified by RP HPLC using a C18 column with a gradient ofH₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 15 mg of the product(154-2). ¹H NMR (300 MHz, CDCl₃) δ 0.04 (s, 9H), 1.15-1.24 (m, 2H), 1.26(t, 3H, J=7 Hz), 1.48 (d, 3H, J=7 Hz), 1.93 (s, 3H), 2.10-2.25 (m, 2H),2.18 (s, 3H), 3.10-3.31 (m, 2H), 3.48 (d, 2H, J=7 Hz), 3.48-3.61 (m,2H), 3.77 (s, 3H), 4.04-4.21 (m, 2H), 4.29-4.40 (m, 2H), 4.81-4.92 (m,1H), 5.13 (s, 2H), 5.64 (t, 1H, J=7 Hz), 8.70-9.11 (m, 3H) ppm; ³¹P(121.4 MHz, CDCl₃) δ 21.9 ppm; MS (m/z) 586.3 [M+H]⁺, 1171.4 [2M+H]⁺.

2-(Hydroxy-{2-[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-ethyl}-phosphinoyloxy)-propionicAcid

A solution of2-[hydroxy-(2-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphinoyloxy]-propionicacid ethyl ester (15 mg, 0.026 mmol) in 10% TFA-CH₂Cl₂ (1 mL) wasstirred at ambient temperature for 10 minutes. The reaction was workedup by removal of the solvent. The residue was dissolved in THF (0.5 mL)and water (0.4 mL) and 1N aqueous NaOH solution (0.1 mL) was added. Thesolution was stirred at room temperature for 20 minutes when it wasacidified with 1N aqueous HCl solution. The resulting solution waspurified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 6.8 mg of the product as a whitesolid. ¹H NMR (300 MHz, CDCl₃) δ 1.38 (d, 3H, J=7 Hz), 1.91 (s, 3H),2.13 (s, 3H), 2.12-2.28 (m, 2H), 3.12-3.33 (m, 2H), 3.41 (d, 2H, J=6Hz), 3.56 (br s, 2H), 3.75 (s, 3H), 4.71-4.88 (m, 1H), 5.16 (s, 2H),5.58-5.71 (m, 1H), 7.88 (br s, 3H), 8.60 (br s, 1H), 8.78 (br s, 1H)ppm; ³¹P (121.4 MHz, CDCl₃) δ 22.0 ppm; MS (m/z) 458.3 [M+H]⁺, 480.3[M+Na]⁺.

Example 175 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

{1-Cyano-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-3-methyl-pent-3-enyl}-phosphonicAcid Diethyl Ester

To a solution of diethyl cyanomethylphosphonate (241 mg, 1.38 mmol) inTHF (1 mL) was added a THF solution of sodium bis(trimethysilyl)amide(1.0 M, 1.13 mL, 1.15 mmol). After stirring for 30 minutes, the solutionwas added dropwise to a solution of6-(4-bromo-3-methyl-but-2-enyl)-7-hydroxy-5-methoxy-4-methyl-3H-isobenzofuran-1-one(100 mg, 0.23 mmol) in THF (1 mL). The resulting mixture was allowed tostir at room temperature for one hour before saturated aqueous ammoniumchloride was added. The reaction mixture was extracted with ethylacetate. The organic layer was dried over sodium sulfate andconcentrated to dryness. The residue was purified by silica gel columnchromatography, affording 110 mg (90%) of the desired product. ¹H NMR(300 MHz, CDCl₃) δ 0.04 (s, 9H), 1.24 (dd, J=7, 8 Hz, 2H), 1.36 (t, 6H),1.86 (s, 3H), 2.17 (s, 3H), 2.43-2.57 (m, 2H), 3.04-3.17 (m, 1H), 3.47(d, J=7.2 Hz, 2H), 3.79 (s, 3H), 4.12-4.37 (m, 6H), 5.13 (s, 2H), 5.44(t, J=7.2 Hz, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 18.18 ppm; MS (m/z) 560[M+Na]⁺.

[1-Cyano-5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicacid diethyl ester

{1-Cyano-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-3-methyl-pent-3-enyl}-phosphonicacid diethyl ester (25 mg, 0.047 mmol) was dissolved in a solution of10% TFA/CH₂Cl₂ (5 mL) and stirred at room temperature for 2 hours. Thereaction mixture was dried under reduced pressure and the product waspurified by RP-HPLC to provide 16 mg (80%) of the desired product as awhite solid. ¹H NMR (300 MHz, CDCl₃) δ 1.38 (t, 6H), 1.86 (s, 3H), 2.15(s, 3H), 2.40-2.58 (m, 2H), 3.01-3.14 (m, 1H), 3.45 (d, J=7.2 Hz, 2H),3.79 (s, 3H), 4.18-4.30 (m, 4H), 5.21 (s, 2H), 5.48 (t, J=7.2 Hz, 1H)ppm; ³¹P (121.4 MHz, CDCl₃) δ 18.09 ppm; MS (m/z) 436 [M−H]⁻, 438[M+H]⁺.

Example 176 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

[1-Cyano-5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-3-methyl-pent-3-enyl]-phosphonicAcid

To a solution of{1-cyano-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-3-methyl-pent-3-enyl}-phosphonicacid diethyl ester (35 mg, 0.065 mmol) in acetonitrile (2 mL) was addedTMSBr (180 μL, 1.38 mmol) and 2,6-lutidine (160 μL, 1.38 mmol). Thereaction solution was allowed stir at room temperature for one hourbefore quenching with MeOH. The reaction mixture was dried under reducedpressure and the residue was purified by RP HPLC using a C18 column witha gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 15 mg(60%) of the desired product. ¹H NMR (300 MHz, CD₃OD) δ 1.86 (s, 3H),2.15 (s, 3H), 2.38-2.57 (m, 2H), 3.17-3.28 (m, 1H), 3.44 (d, J=7.2 Hz,2H), 3.80 (s, 3H), 5.25 (s, 2H), 5.47 (t, J=7.2 Hz, 1H) ppm; ³¹P (121.4MHz, CD₃OD) δ 15.28 ppm; MS (m/z) 380 [M−H]⁻, 382 [M+H]⁺.

Example 177 Preparation of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedbelow.

{1-Cyano-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-1,3-dimethyl-pent-3-enyl}-phosphonicAcid Diethyl Ester

To a solution of{1-cyano-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-3-methyl-pent-3-enyl}-phosphonicacid diethyl ester (45 mg, 0.084 mmol) in THF (0.5 mL) was added sodiumbis(trimethysilyl)amide (1.0 M, 1.13 mL, 1.15 mmol). After stirring for20 minutes, iodomethane (52 μL, 0.84 mmol) was added dropwise and theresulting mixture was allowed to stir at room temperature for 2 hours.The reaction mixture was quenched with saturated aqueous ammoniumchloride and extracted with ethyl acetate. The organic layer was driedover sodium sulfate and concentrated to dryness. The residue waspurified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to afford 6.6 mg (23%) of the desiredproduct. ¹H NMR (300 MHz, CDCl₃) δ 0.00 (s, 9H), 1.16 (dd, J=7, 8 Hz,2H), 1.31 (t, 6H), 1.38 (d, 3H), 1.92 (s, 3H), 2.17 (s, 3H), 2.23 (m,1H), 2.65 (m, 1H), 3.30-3.42 (m, 2H), 3.73 (s, 3H), 4.14-4.27 (m, 6H),5.08 (s, 2H), 5.28 (t, J=7.2 Hz, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 22.26ppm; MS (m/z) 574 [M+Na]⁺.

[1-Cyano-5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-1,3-dimethyl-pent-3-enyl]-phosphonicAcid

To a solution of{1-cyano-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-1,3-dimethyl-pent-3-enyl}-phosphonicacid diethyl ester (18 mg, 0.04 mmol) in DMF (0.5 mL) and DCM (0.5 mL)was added TMSBr (51 μL, 0.4 mmol) and 2,6-lutidine (46 μL, 0.4 mmol).The reaction solution was allowed stir at room temperature overnightbefore quenching with MeOH. The reaction mixture was dried under reducedpressure and the residue was purified by RP HPLC using a C18 column witha gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 4.5 mg(33%) of the desired product. ¹H NMR (300 MHz, CD₃OD) δ 1.37 (d, 3H),1.87 (s, 3H), 2.13 (s, 3H), 2.26 (m, 1H), 2.64 (m, 1H), 3.39 (m, 2H),3.75 (s, 3H), 5.18 (s, 2H), 5.34 (m, 1H) ppm; ³¹P (121.4 MHz, CD₃OD) δ21.47 ppm; MS (m/z) 422 [M−H]⁻, 424 [M+H]⁺.

Example 178 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

2-Ethyl-4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enal

A solution of[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde(1.5 g, 4.46 mmol) in toluene (14 mL) was heated at 100° C. with2-(triphenyl-phosphanylidene)-butyraldehyde (1.68 g, 5.35 mmol)overnight. A second portion of2-(triphenyl-phosphanylidene)-butyraldehyde (495 mg, 1.49 mmol) wasadded and the reaction mixture was heated for an additional day. Afterconcentration, the residue was purified by silica gel chromatography toprovide 1.3 g (83%) of the desired product as oil. ¹H NMR (300 MHz,CDCl₃) δ 0.01 (s, 9H), 1.03 (t, 3H), 1.10-1.21 (m, 2H), 2.15 (s, 3H),2.15-2.44 (m, 2H), 3.67-3.76 (m, 2H), 3.74 (s, 3H), 4.31-4.36 (m, 2H),5.10 (s, 2H), 6.34-6.38 (m, 1H), 9.28 (s, 1H) ppm.

6-(3-Hydroxymethyl-pent-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

A solution of2-ethyl-4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enal(1.3 g, 3.30 mmol) in methanol (10 mL) and THF (10 mL) was cooled to 0°C. A solution of CeCl₃ (8.25 mL, 0.4M, MeOH: H₂O, 9:1) was added,followed by LiBH₄ (1.66 mL, 3.30 mmol of a 2M solution in THF). The icebath was removed and the reaction mixture was allowed to warm to roomtemperature. The reaction mixture was stirred for an additional 40minutes whereupon TLC indicated complete consumption of startingaldehyde. The reaction was worked up by addition of aqueous 1N HCl andthe product was extracted with EtOAc. The organic layer was washed withsaturated aqueous sodium bicarbonate solution and brine. The organiclayer was concentrated under reduced pressure and the residue waspurified by silica gel chromatography to provide 948 mg (73%) of theproduct as colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 0.00 (s, 9H), 1.07(t, 3H), 1.20 (dd, 2H, J=7, 8 Hz), 2.13 (s, 3H), 2.38-2.50 (m, 2H), 3.77(s, 3H), 3.99 (s, 2H), 4.27 (dd, 2H, J=7, 8 Hz), 5.08 (s, 2H), 5.34 (t,J=7.2 Hz, 1H) ppm.

6-(3-Bromomethyl-pent-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

Polymer-supported triphenylphosphine (3 mmol/g, 0.66 g) was soaked indichloromethane (6 mL) for 1 hour.6-(3-Hydroxymethyl-pent-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(260 mg, 0.66 mmol) and carbon tetrabromide (657 mg, 1.98 mmol) wereadded sequentially and the mixture was shaken for 1 h at roomtemperature. The mixture was filtered and the filtrate was concentrated.The residue was purified by silica gel chromatography to provide 233 mg(77%) of the product as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 0.00(s, 9H), 1.08 (t, 3H), 1.20 (dd, 2H, J=7, 8 Hz), 2.14 (s, 3H), 2.35-2.43(m, 2H), 3.44 (d, J=7.2, 2H), 3.73 (s, 3H), 3.95 (s, 2H), 4.27 (dd, 2H,J=7, 8 Hz), 5.08 (s, 2H), 5.53 (t, J=7.2 Hz, 1H) ppm.

[2-Ethyl-4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-but-2-enyl]-phosphonicacid

A solution of6-(3-bromomethyl-pent-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(230 mg, 0.5 mmol) in trimethylphosphite (1.5 mL, 12.75 mmol) was heatedto 100° C. for 4 hours. The reaction was worked up by removal of excesstrimethylphosphite under reduced pressure. The residue was dissolved inacetonitrile (1 mL) and TMSBr (646 L, 5.0 mmol) and 2,6-lutidine (580μL, 5.0 mmol) were added at 0° C. The reaction solution was allowed towarm to room temperature and stirred for 4 hours. The reaction wascooled to 0° C. and quenched with addition of MeOH. The reaction mixturewas dried under reduced pressure and the residue was purified by RP HPLCusing a C18 column with a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1%TFA to provide 77 mg (58%) of the product. ¹H NMR (300 MHz, CD₃₀D) δ1.08 (t, 3H), 2.16 (s, 3H), 2.43 (m, 2H), 2.48 (d, 2H, J=22 Hz), 3.46(t, 2H, J=6 Hz), 3.79 (s, 3H), 5.25 (s, 2H), 5.38 (q, 1H, J=7 Hz) ppm.;³¹P (121.4 MHz, CD₃₀D) δ 25.65 ppm.; MS (m/z) 355 [M−H]⁻, 357 [M+H]⁺

Example 179 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

{1-Cyano-3-ethyl-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-pent-3-enyl}-phosphonicAcid Diethyl Ester

To a solution of diethyl cyanomethylphosphonate (233 mg, 1.32 mmol) inTHF (1 mL) was added a THF solution of sodium bis(trimethysilyl)amide(1.0 M, 1.21 mL, 1.21 mmol). After stirring for 30 minutes, the solutionwas added dropwise to a solution of6-(3-bromomethyl-pent-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(100 mg, 0.22 mmol) in THF (1 mL). The resulting mixture was allowed tostir at room temperature overnight before saturated aqueous ammoniumchloride was added. The reaction mixture was extracted with ethylacetate. The organic layer was dried over sodium sulfate andconcentrated to dryness. The residue was purified by preparativereverse-phase HPLC, affording 51 mg (42%) of the desired product. ¹H NMR(300 MHz, CDCl₃) δ 0.04 (s, 9H), 1.07 (t, 3H), 1.24 (dd, 2H, J=7, 8 Hz),1.36 (t, 6H), 2.12 (m, 1H), 2.18 (s, 3H), 2.35-2.47 (m, 2H), 2.67 (m,1H), 3.00-3.14 (m, 1H), 3.44 (d, J=7.2, 2H), 3.79 (s, 3H), 4.12-4.37 (m,6H), 5.13 (s, 2H), 5.38 (t, J=7.2 Hz, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ18.26 ppm; MS (m/z) 574 [M+Na]⁺.

[1-Cyano-3-ethyl-5-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-pent-3-enyl]-phosphonicAcid

{1-Cyano-3-ethyl-5-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-pent-3-enyl}-phosphonicacid diethyl ester (19.5 mg, 0.035 mmol) was dissolved in a solution of10% TFA/CH₂Cl₂ (2 mL) and stirred at room temperature for 10 minutes.The reaction mixture was dried under reduced pressure and purified byRP-HPLC to provide 9.5 mg (61%) of the desired product. This materialwas dissolved in DMF (0.5 mL) and DCM (0.5 mL) and TMSBr (27 μL, 0.2mmol) and 2,6-lutidine (23 μL, 0.2 mmol) were added. The reactionsolution was allowed stir at room temperature overnight before quenchingwith MeOH. The reaction mixture was dried under reduced pressure and theresidue was purified by RP HPLC using a C18 column with a gradient ofH₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 5.1 mg (65%) of thedesired product as a white solid. ¹H NMR (300 MHz, CD₃OD) δ 1.10 (t,3H), 2.16 (s, 3H), 2.23-2.52 (m, 3H), 2.67 (m, 1H), 3.05-3.20 (m, 1H),3.48 (d, J=7.2, 2H), 3.81 (s, 3H), 5.26 (s, 2H), 5.43 (t, J=7.2 Hz, 1H)ppm; ³¹P (121.4 MHz, CD₃OD) δ 14.18 ppm; MS (m/z) 394 [M−H]⁻, 396[M+H]⁺.

Example 180 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

{2-Ethyl-4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enyloxymethyl}-phosphonicAcid Diisopropyl Ester

To a solution of bromomethylphosphonate diisopropyl ester (680 mg, 2.62mmol) and6-(3-hydroxymethyl-pent-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(688 mg, 1.75 mmol) in DMF (3 mL) was added lithium t-butoxide (1.0M inTHF; 2.6 mL). The reaction was heated at 70° C. for 2 hours. Aftercooling to ambient temperature, more bromomethylphosphonate diisopropylester (680 mg, 2.62 mmol) and lithium t-butoxide (1.0M in THF; 2.6 mL)were added. The reaction mixture was heated at 70° C. for a furtherhour, cooled, poured into a solution of lithium chloride (5% aqueous)and extracted with ethyl acetate. The organic extract was dried and theproduct was purified by chromatography on silica gel, eluting withhexane-ethyl acetate to provide 347 mg (35%) of the product as acolorless oil. ¹H NMR (300 MHz, CDCl₃) δ 0.04 (s, 9H), 1.09 (t, 3H,J=7.5 Hz), 1.20-1.26 (m, 2H), 1.31 (t, 12H, J=6 Hz), 2.18 (s, 3H), 2.29(q, 2H, J=7.5 Hz), 3.5 (m, 2H), 3.59 (d, 2H, J=8.7 Hz), 3.78 (s, 3H),3.98 (s, 2H), 4.28-4.35 (m, 2H), 4.6-4.8 (m, 2H), 5.13 (s, 2H), 5.4 (t,1H, J=7 Hz) ppm; ³¹P (121.4 MHz, CDCl₃) δ 20.26 ppm; MS (m/z) 593.3[M+Na]⁺.

[2-Ethyl-4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-but-2-enyloxymethyl]-phosphonicAcid

To a solution of{2-ethyl-4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enyloxymethyl}-phosphonicacid diisopropyl ester (347 mg, 0.61 mmol) in acetonitrile (5 mL) wasadded 2,6-lutidine (0.71 mL, 6.1 mmol) and bromotrimethylsilane (0.786mL, 6.1 mmol). The mixture was stirred at room temperature for 3 hours,quenched with methanol (5 mL), concentrated, and partitioned betweenethyl acetate and 1N HCl (aqueous). The organic layer was concentratedto give the free phosphonic acid as a colorless oil (205 mg, 70%). Thismaterial (20 mg) was dissolved in a solution of trifluoroacetic acid(0.3 mL) and dichloromethane (2.7 mL) and stirred for 30 minutes atambient temperature. After concentration, the residue was purified by RPHPLC using a C18 column with a gradient of H₂O, 0.1% TFA-acetonitrile,0.1% TFA to provide the product, after lyophilization, as a white solid(10 mg). ¹H NMR (300 MHz, CDCl₃) δ 1.007 (t, 3H, J=7.5 Hz), 2.13 (s,3H), 2.32 (q, 2H, J=7.5 Hz), 3.41 (d, 2H, J=6.3 Hz), 3.56 (d, 2H, J=9Hz), 3.75 (s, 3H), 3.95 (s, 2H), 5.16 (s, 2H), 5.43 (t, 1H, J=6.3 Hz)ppm; ³¹P (121.4 MHz, CDCl₃) δ 22.8 ppm; MS (m/z) 385.2 [M−H]⁺, 387.1[M+H]⁺.

Example 181 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

6-Allyloxy-3-methyl-4-trifluoromethanesulfonyloxy-phthalic Acid DimethylEster

To a solution of 6-allyloxy-4-hydroxy-3-methyl-phthalic acid dimethylester (8.06 g, 28.8 mmol) [synthesized according to: J. W. Patterson,Tetrahedron, 1993, 49, 4789-47981 and pyridine (11.4 g, 144.0 mmol) indichloromethane (DCM) (20 mL) at 0° C. was added triflic anhydride(12.19 g, 43.2 mmol). The reaction was stirred at 0° C. for 2 hrs afterwhich additional triflic anhydride (3 mL) was added. Stirring at 0° C.was continued for an additional hour. The reaction mixture was pouredinto a mixture of DCM and HCl (1N). The layers were separated and theaqueous layer was extracted with DCM. The combined organic layers weredried over sodium sulfate. Filtration and evaporation of solvents invacuo yielded a crude product, which was purified by silica gelchromatography to provide 8.39 g of the product as an oil. ¹H NMR (300MHz, CDCl₃): δ=2.32 (s, 3H), 3.89 (s, 6H), 4.60 (m, 2H), 5.33 (d, J=9.3Hz, 1H), 5.41 (d, J=18.6 Hz, 1H), 5.95 (m, 1H), 6.95 (s, 1H) ppm; ¹⁹FNMR (282 MHz, CDCl₃): δ=−74 ppm.

6-Hydroxy-3-methyl-4-trifluoromethanesulfonyloxy-phthalic Acid DimethylEster

To a solution of6-allyloxy-3-methyl-4-trifluoromethanesulfonyloxy-phthalic acid dimethylester (8.39 g, 20.3 mmol) in toluene (20 mL) was addedtetrakistriphenylphosphine palladium (0.47 g, 0.40 mmol) anddiethylamine (2.97 g, 40.86 mmol) at room temperature under anatmosphere of nitrogen. Stirring at room temperature was continued untilall starting material was consumed. The crude reaction mixture waspartitioned between diethyl ether and HCl (0.1 N). The organic layer waswashed with brine and dried over sodium sulfate. Filtration andevaporation of solvents in vacuo yielded a crude material, which waspurified by silica gel chromatography to provide 4.16 g (55%) of thedesired product as an off-white solid. ¹H NMR (300 MHz, CDCl₃): δ=2.20(s, 3H), 3.93 (s, 3H), 3.95 (s, 3H), 7.01 (s, 1H) ppm; ¹⁹F NMR (282 MHz,CDCl₃): δ=−74 ppm.

6-Hydroxy-3-methyl-4-vinyl-phthalic Acid Dimethyl Ester

To a solution of6-hydroxy-3-methyl-4-trifluoromethanesulfonyloxy-phthalic acid dimethylester (2.17 g, 5.85 mmol) in N-methylpyrrolidinone (15 mL) was addedlithium chloride (743 mg, 17.5 mmol) and triphenylarsine (179 mg, 0.585mmol). Tributylvinyltin (2.04 g, 6.43 mmol) was added followed bytris(tribenzylideneacetone)dipalladium(0)-chloroform adduct (90 mg,0.087 mmol). The reaction was placed under an atmosphere of nitrogen andheated at 60° C. for 18 hrs. The reaction was cooled to room temperatureand poured onto a mixture of ice (20 g), EtOAc (40 mL), and potassiumfluoride (1 g). Stirring was continued for 1 hr. The aqueous layer wasextracted with EtOAc and the organic extracts filtered through Celite.The combined organic layers were washed with water and dried over sodiumsulfate. Filtration and evaporation of solvents in vacuo yielded a crudematerial, which was purified by silica gel chromatography to provide1.27 g (87%) of the product as an off-white solid. ¹H NMR (300 MHz,CDCl₃): δ=2.16 (s, 3H), 3.91 (s, 3H), 3.92 (s, 3H), 5.46 (dd, J=11.1,1.2 Hz, 1H), 5.72 (dd, J=17.1, 0.9 Hz, 1H), 6.86 (dd, J=17.1, 11.1 Hz,1H), 7.14 (s, 1H), 10.79 (s, 1H) ppm.

4-Ethyl-6-hydroxy-3-methyl-phthalic Acid Dimethyl Ester

6-Hydroxy-3-methyl-4-vinyl-phthalic acid dimethyl ester (1.27 g, 5.11mmol) was dissolved in benzene (10 mL) and EtOAc (10 mL).Tristriphenylphosphine rhodium chloride (150 mg) was added and thereaction was placed under an atmosphere of hydrogen. Stirring at roomtemperature was continued. After 14 hrs, the solvents were removed invacuo and the crude material was purified by silica gel chromatographyto provide 1.14 g (88%) of the desired product as an off-white solid. ¹HNMR (300 MHz, CDCl₃): δ=1.19 (t, J=7.8 Hz, 3H), 2.10 (s, 3H), 2.60 (q,J=7.8 Hz, 2H), 3.89 (s, 6H), 6.87 (s, 1H), 10.79 (s, 1H) ppm.

6-Allyloxy-4-ethyl-3-methyl-phthalic Acid Dimethyl Ester

4-Ethyl-6-hydroxy-3-methyl-phthalic acid dimethyl ester (1.01 g, 4.02mmol) was dissolved in DMF (5 mL). Potassium carbonate (3.33 g, 24.14mmol) was added, followed by allylbromide (2.92 g, 24.14 mmol). Thesuspension was heated at 60° C. After 14 hrs, the reaction was cooled toroom temperature and filtered. The solvents were removed in vacuo andthe crude material was purified by silica gel chromatography to provide0.976 g (83%) of the desired product as a colorless oil. ¹H NMR (300MHz, CDCl₃): δ=1.16 (t, J=7.2 Hz, 3H), 2.20 (s, 3H), 2.62 (q, J=7.2 Hz,2H), 3.83 (s, 3H), 3.84 (s, 3H), 4.57 (m, 2H), 5.26 (dd, J=9.3, 1.5 Hz,1H), 5.41 (dd, J=13.5, 1.5 Hz, 1H), 5.98 (m, 1H), 6.82 (s, 1H) ppm.

4-Allyl-5-ethyl-3-hydroxy-6-methyl-phthalic Acid Dimethyl Ester

6-Allyloxy-4-ethyl-3-methyl-phthalic acid dimethyl ester (1.25 g, 4.28mmol) was heated at 210° C. under an atmosphere of nitrogen. After 14hrs, the reaction was cooled to room temperature. The crude material waspurified by silica gel chromatography to provide 0.971 g (77%) of thedesired product as a colorless oil. ¹H NMR (300 MHz, CDCl₃): δ=1.14 (t,J=7.8 Hz, 3H), 2.17 (s, 3H), 2.68 (q, J=7.8 Hz, 2H), 3.49 (m, 2H), 3.86(s, 3H), 3.89 (s, 3H), 4.89-5.01 (m, 2H), 5.93 (m, 1H), 11.22 (s, 1H)ppm.

6-Allyl-5-ethyl-7-hydroxy-4-methyl-3H-isobenzofuran-1-one

4-Allyl-5-ethyl-3-hydroxy-6-methyl-phthalic acid dimethyl ester (0.971g, 3.32 mmol) was dissolved in MeOH (8 mL) at room temperature. Asolution of sodium hydroxide (0.798 g, 19.95 mmol) in water (10 mL) wasadded and the suspension was heated at 55° C. After 16 hrs, the reactionwas cooled to room temperature and washed with diethyl ether. Theaqueous layer was acidified (1N HCl) and the suspension was extractedwith EtOAc. The combined organic layers were dried over sodium sulfate.Filtration and evaporation of solvents in vacuo yielded the desired bisacid as a white solid (0.846 g, 98%, M⁺=263). The bis acid was dissolvedin acetic acid (6 mL) and HCl (conc., 1.5 mL). The reaction was heatedat 80° C. Zn dust (0.635 g, 9.72 mmol, each) was added in portions everyhour for 7 hours. Stirring at 80° C. was continued for additional 10hours. The reaction was cooled to room temperature and water was added.The resultant suspension was extracted with EtOAc. The combined organicextracts were washed with sodium bicarbonate solution and dried oversodium sulfate. Filtration and evaporation of solvents in vacuo yieldedthe crude product, which was purified by silica gel chromatography toprovide 0.375 g (50%) of the product as a white solid. ¹H NMR (300 MHz,CDCl₃): δ=1.14 (t, J=7.5 Hz, 3H), 2.18 (s, 3H), 2.71 (q, J=7.5 Hz, 2H),3.49 (m, 2H), 4.95 (d, J=17.1 Hz, 1H), 5.02 (d, J=10.2 Hz, 1H), 5.23 (s,2H), 5.98 (m, 1H), 7.66 (s, 1H) ppm.

6-Allyl-5-ethyl-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

To a solution of6-allyl-5-ethyl-7-hydroxy-4-methyl-3H-isobenzofuran-1-one (199 mg, 0.857mmol), PPh₃ (337 mg, 1.286 mmol), and 2-trimethylsilylethanol in THF (3mL) at 0° C. was added diisopropyl azodicarboxylate (259 mg, 1.286mmol). The resulting yellow solution was allowed to warm to roomtemperature and stirred for one hour. The solvent was removed in vacuoand the crude material was dissolved in diethyl ether (3 mL). Hexanes(1.5 mL) were added. Triphenylphosphine oxide was removed by filtrationand the filtrate was concentrated and purified by silica gelchromatography to provide the desired product (261 mg, 92%) as a clearoil. ¹H NMR (300 MHz, CDCl₃): δ=0.04 (s, 9H), 1.15 (t, J=7.8 Hz, 3H),1.25 (m, 2H), 2.20 (s, 3H), 2.73 (q, J=7.8 Hz, 2H), 3.54 (m, 2H), 4.28(m, 2H), 4.95 (d, J=17.1 Hz, 1H), 5.02 (d, J=10.2 Hz, 1H), 5.15 (s, 2H),5.95 (m, 1H) ppm.

[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde

A solution of6-allyl-5-ethyl-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(261 mg, 0.788 mmol) in MeOH (5 mL), CH₂Cl₂ (5 mL) and pyridine (50 μL)was cooled to −78° C. using a dry ice/acetone bath according to theprocedure of Smith, D. B. et al., J. Org. Chem., 1996, 61, 6, 2236. Astream of ozone was bubbled through the reaction via a gas dispersiontube until the reaction became blue in color (15 minutes). The ozoneline was replaced with a stream of nitrogen and bubbling continued foranother 15 minutes, by which time the blue color had disappeared. Tothis solution at −78° C. was added thiourea (59.9 mg, 0.788 mmol) in oneportion, and the cooling bath was removed. The reaction was allowed towarm to room temperature and stirred for 15 hours. The reaction mixturewas filtered and then partitioned between CH₂Cl₂ and water. The aqueouslayer was extracted with CH₂Cl₂ one more time and the organic extractswere combined, washed with aqueous 1N HCl, saturated NaHCO₃ and brineand dried over sodium sulfate. Filtration and evaporation of solvents invacuo yielded the crude product, which was purified by silica gelchromatography to afford 181 mg (69%) of the product as a white solid.¹H NMR (300 MHz, CDCl₃): δ=0.04 (s, 9H), 1.11 (t, J=7.5 Hz, 3H), 1.19(m, 2H), 2.21 (s, 3H), 2.66 (q, J=7.5 Hz, 2H), 3.90 (s, 2H), 4.36 (m,2H), 5.18 (s, 2H), 9.71 (s, 1H) ppm.

4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal

[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde(90 mg, 0.269 mmol) and 2-(triphenyl-phosphorylidene)-propionaldehyde(72.9 mg, 0.23 mmol) in toluene (3 mL) were heated at 100° C. After 15hours, a second portion of 2-(triphenyl-phosphanylidene)-propionaldehyde(33 mg, 0.11 mmol) was added and the reaction mixture was heated foradditional 9 hours. The toluene was removed in vacuo, and the residuewas purified by silica gel chromatography to provide 77.6 mg (77%) ofthe desired product as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃):δ=0.03 (s, 9H), 1.15 (t, J=7.5 Hz, 3H), 1.21 (m, 2H), 1.93 (s, 3H), 2.21(s, 3H), 2.71 (q, J=7.5 Hz, 2H), 3.82 (d, J=6.9 Hz, 2H), 4.34 (m, 2H),5.18 (s, 2H), 6.38 (m, 1H), 9.35 (s, 1H) ppm.

5-Ethyl-6-(4-hydroxy-3-methyl-but-2-enyl)-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(77.6 mg, 0.207 mmol) was dissolved in MeOH (4 mL). A solution of CeCl₃(51.1 mg, 0.207 mmol) in MeOH/water (9/1, 0.66 mL) was added and thesolution was cooled to 0° C. A solution of lithium borohydride in THF(2M, 0.105 mL) was added dropwise. After 15 minutes, the reaction wasquenched with 1N HCl (0.5 mL). The MeOH was removed in vacuo and thecrude material was partitioned between DCM and water. The aqueous layerwas extracted with DCM and the combined organic layers were washed withsodium bicarbonate solution and dried over sodium sulfate. Filtrationand evaporation of solvents yielded a crude oil, which was purified bysilica gel chromatography to provide 57.2 mg (73%) of the desiredproduct. ¹H NMR (300 MHz, CDCl₃): δ=0.04 (s, 9H), 1.15 (t, J=7.8 Hz,3H), 1.26 (m, 2H), 1.86 (s, 3H), 2.19 (s, 3H), 2.72 (q, J=7.8 Hz, 2H),3.52 (d, J=6.3 Hz, 2H), 3.99 (s, 2H), 4.34 (m, 2H), 5.14 (s, 2H), 5.32(m, 1H) ppm.

6-(4-Bromo-3-methyl-but-2-enyl)-5-ethyl-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

5-Ethyl-6-(4-hydroxy-3-methyl-but-2-enyl)-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(57.2 mg, 0.152 mmol) was dissolved in DCM (3.5 mL). Polymer-boundtriphenylphosphine (3 mmol/g, 152.1 mg) was added and the mixture wasmechanically stirred at room temperature. Carbon tetrabromide (151.3 mg,0.456 mmol) was added and the solution was stirred at room temperature.After 2 hrs, the reaction was filtered and the solvent was removed invacuo. The crude material was purified by silica gel chromatography toprovide 58.0 mg (87%) of the desired product. ¹H NMR (300 MHz, CDCl₃):δ=0.04 (s, 9H), 1.15 (t, J=7.8 Hz, 3H), 1.25 (m, 2H), 1.95 (s, 3H), 2.20(s, 3H), 2.70 (q, J=7.8 Hz, 2H), 3.52 (d, J=6.3 Hz, 2H), 3.94 (s, 2H),4.28 (m, 2H), 5.14 (s, 2H), 5.50 (m, 1H) ppm.

{4-[6-Ethyl-7-methyl-3-oxo-4-hydroxy-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicAcid

A solution of4-[6′-ethyl-7′-methyl-3′-oxo-4′-(2″-trimethylsilanyl-ethoxy)-1′,3′-dihydro-isobenzofuran-5′-yl]-2-methyl-but-2-enylbromide (58 mg, 0.132 mmol) in trimethylphosphite (0.8 mL) was heated at110° C. After 2 hrs the reaction was complete. The reaction was cooledto room temperature and the excess trimethylphosphite was removed invacuo. The crude material was used in the next step without furtherpurification. The crude product of the Arbuzov reaction was dissolved inMeCN (0.8 mL). Trimethylsilyl bromide (202.2 mg, 1.321 mmol) was addedand the reaction was stirred at room temperature. After 15 minutes,lutidine (155.7 mg, 1.453 mmol) was added and stirring at roomtemperature was continued. After 2 hrs additional trimethylsilyl bromide(202.2 mg, 1.321 mmol) was added and stirring at room temperature wascontinued. After 4 hrs the reaction was quenched with MeOH (2 mL). Thesolvents were evaporated in vacuo and the crude material was purified byRP-HPLC (eluent: water/MeCN). The product-containing fractions werecombined and lyophilized to yield 2.3 mg (5.1%) of the free phosphonicacid. ¹H NMR (300 MHz, DMSO-d6): δ=1.07 (t, J=7.5 Hz, 3H), 1.84 (s, 3H),2.14 (s, 3H), 2.64 (q, J=7.5 Hz, 2H), 3.34 (m, 4H), 5.06 (m, 1H), 5.25(s, 2H) ppm; ³¹P NMR (121 MHz, DMSO-d6): δ=22.19 ppm; MS=341 [M⁺+1].

Example 182 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

[2-Ethyl-4-[6-ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enal

[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-acetaldehyde(90 mg, 0.269 mmol) and 2-(triphenyl-phosphorylidene)-butyraldehyde(98.4 mg, 0.296 mmol) in toluene (3 mL) were heated at 100° C. After 15hours, a second portion of 2-(triphenyl-phosphanylidene)-butyraldehyde(98.4 mg, 0.296 mmol) was added and the reaction mixture was heated foradditional 33 hours. After concentration, the residue was purified bysilica gel chromatography to provide 50.3 mg (48%) of the desiredproduct as a pale yellow oil.

5-Ethyl-6-(3-hydroxymethyl-pent-2-enyl)-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

2-Ethyl-4-[6-ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enal(50.3 mg, 0.129 mmol) was dissolved in MeOH (3 mL). A solution of CeCl₃(31.9 mg, 0.129 mmol) in MeOH/water (9/1, 0.66 mL) was added and thesolution was cooled to 0° C. A solution of lithium borohydride in THF(2M, 0.065 mL) was added dropwise. After 10 minutes, the reaction wasquenched with 1N HCl (0.5 mL). The methanol was removed in vacuo and thecrude material was partitioned between DCM and water. The aqueous layerwas extracted with DCM and the combined organic layers were washed withsodium bicarbonate solution and were dried over sodium sulfate.Filtration and evaporation of solvents in vacuo yielded a crude oil,which was purified by silica gel chromatography to provide 35.4 mg (70%)of the desired product. ¹H NMR (300 MHz, CDCl₃): δ=0.04 (s, 9H),1.10-1.19 (m, 6H), 1.26 (m, 2H), 2.19 (s, 3H), 2.32 (q, J=7.5 Hz, 2H),2.72 (q, J=7.5 Hz, 2H), 3.54 (d, J=6.6 Hz, 2H), 4.05 (s, 2H), 4.26 (m,2H), 5.14 (s, 2H), 5.27 (m, 1H) ppm.

6-(3-Bromomethyl-pent-2-enyl)-5-ethyl-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one

5-Ethyl-6-(3-hydroxymethyl-pent-2-enyl)-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(35.4 mg, 0.090 mmol) was dissolved in DCM (3.0 mL). Polymer-boundtriphenylphosphine (3 mmol/g, 90.7 mg) was added and the mixture wasmechanically stirred at room temperature. Carbon tetrabromide (90.2 mg,0.272 mmol) was added and the solution was stirred at room temperature.After 2 hrs, the reaction was filtered and the solvent was removed invacuo. The crude material was purified by silica gel chromatography toprovide 32.0 mg (78%) of the desired product. The material was used inthe next step without further characterization.

[2-Ethyl-4-(6-ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-but-2-enyl]-phosphonicAcid

A solution of6-(3-bromomethyl-pent-2-enyl)-5-ethyl-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(32 mg, 0.070 mmol) in trimethylphosphite (0.8 mL) was heated at 110° C.After 2 hrs the reaction was complete. The reaction was cooled to roomtemperature and the excess trimethylphosphite was removed in vacuo. Thecrude material was used in the next step without further purification.The crude product of the Arbuzov reaction was dissolved in MeCN (0.8mL). Trimethylsilyl bromide (108.0 mg, 0.706 mmol) was added and thereaction was stirred at room temperature. After 2 hrs a second batch oftrimethysilyl bromide (108.0 mg, 0.706 mmol) was added. After 3 hrs thereaction was quenched with MeOH (2 mL). The solvents were evaporated invacuo and the crude material was purified by RP-HPLC (eluent:water/MeCN). The product-containing fractions were combined andlyophilized to yield 15.7 mg (63%) of the product. ¹H NMR (300 MHz,DMSO-d6): δ=0.98-1.09 (m, 6H), 2.10 (s, 3H), 2.30 (m, 2H), 2.64 (q,J=7.5 Hz, 2H), 3.38 (m, 4H), 5.03 (m, 1H), 5.25 (s, 2H) ppm; ³¹P NMR(121 MHz, DMSO-d6): δ=22.26 ppm; MS=355 [M⁺+1].

Example 183 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

(2-{4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicAcid Diethyl Ester

4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(19.7 mg, 0.052 mmol) and aminoethylphosphonic acid diethylester oxalatesalt (15.6 mg, 0.057 mmol) were dissolved in DMF (0.5 mL). Acetic acid(15.7 mg, 0.263 mmol) was added, followed by sodiumtriacetoxyborohydride (22.3 mg, 0.105 mmol). After 4 hrs, the crudereaction mixture was purified by RP-HPLC (eluent: water/MeCN) to provide27.7 mg (97%) of the desired product after lyophilization. ¹H NMR (300MHz, CDCl₃): δ=0.04 (s, 9H), 1.14 (t, J=7.5 Hz, 3H), 1.26 (m, 2H), 1.30(t, J=7.2 Hz, 6H), 1.95 (s, 3H), 2.19 (s, 3H), 2.23 (m, 2H), 2.68 (q,J=7.5 Hz, 2H), 3.18 (m, 2H), 3.53 (s, 2H), 4.13 (m, 4H), 4.28 (m, 2H),5.15 (s, 2H), 5.51 (m, 1H) ppm; ³¹P NMR (121 MHz, CDCl₃): δ=27.39 ppm;MS=540 [M⁺+1].

{2-[4-(6-Ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-ethyl}-phosphonicAcid

(2-{4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (27.7 mg, 0.051 mmol) was dissolved in DMF (0.5 mL)and DCM (0.5 mL). Trimethylsilyl bromide (78.3 mg, 0.512 mmol) was addedand the reaction was stirred at room temperature. After 20 hrs thereaction was quenched with MeOH (0.3 mL). The solvents were evaporatedin vacuo and the crude material was purified by RP-HPLC (eluent:water/MeCN). The product-containing fractions were combined andlyophilized to yield 14.2 mg (57%) of the free phosphonic acid [MS: 484M⁺+1].

The material was dissolved in DCM (0.5 mL). TFA (0.05 mL) was added andstirring at room temperature was continued. After 20 minutes, thesolvents were removed in vacuo and the crude material was purified byRP-HPLC (eluent: water/MeCN*0.1% TFA). The product-containing fractionswere combined and lyophilized to yield 7.6 mg (52%) of the product asthe TFA salt. ¹H NMR (300 MHz, DMSO-d6): δ=1.07 (t, J=7.5 Hz, 3H), 1.84(s, 3H), 1.90 (m, 2H), 2.11 (s, 3H), 2.63 (q, J=7.5 Hz, 2H), 2.99 (m,2H), 3.43 (d, J=6.3 Hz, 2H), 3.51 (s, 2H), 5.26 (s, 2H), 5.45 (m, 1H)ppm; ³¹P NMR (121 MHz, DMSO-d6): δ=20.02 ppm; MS=384 [M⁺+1].

(2-{2-Ethyl-4-[6-ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enylamino}-ethyl)-phosphonicAcid Diethyl Ester

2-Ethyl-4-[6-ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enal(26.6 mg, 0.068 mmol) and aminoethylphosphonic acid diethylester oxalatesalt (20.4 mg, 0.075 mmol) were dissolved in DMF (0.8 mL). Acetic acid(20.5 mg, 0.342 mmol) was added, followed by sodiumtriacetoxyborohydride (27.6 mg, 0.137 mmol). After 8 hrs, the crudereaction mixture was purified by RP-HPLC (eluent: water/MeCN) to provide24.9 mg (65%) of the desired product after lyophilization. ¹H NMR (300MHz, CDCl₃): δ=0.05 (s, 9H), 1.10-1.24 (m, 8H), 1.35 (t, J=7.5 Hz, 6H),2.19 (s, 3H), 2.23 (m, 2H), 2.35 (q, J=7.8 Hz, 2H), 2.70 (q, J=7.2 Hz,2H), 3.25 (m, 2H), 3.56 (m, 4H), 4.15 (m, 4H), 4.29 (m, 2H), 5.15 (s,2H), 5.47 (m, 1H) ppm; ³¹P NMR (121 MHz, CDCl₃): δ=27.71 ppm; MS=554[M⁺+1].

{2-[2-Ethyl-4-(6-ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-but-2-enylamino]-ethyl}-phosphonicAcid

(2-{2-Ethyl-4-[6-ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-but-2-enylamino}-ethyl)-phosphonicacid diethyl ester (24.9 mg, 0.045 mmol) was dissolved in DMF (0.5 mL)and DCM (0.5 mL). Trimethylsilyl bromide (68.7 mg, 0.449 mmol) was addedand the reaction was stirred at room temperature. After 20 hrs thereaction was quenched with MeOH (0.15 mL). The solvents were evaporatedin vacuo and the crude material was purified by RP-HPLC (eluent:water/MeCN). The product-containing fractions were combined andlyophilized to yield 8.0 mg of the free phosphonic acid [MS: 498 M⁺+1].

This material was dissolved in DCM (0.5 mL). TFA (0.05 mL) was added,and stirring at room temperature was continued. After 20 minutes, thesolvents were removed in vacuo and the crude material was purified byRP-HPLC (eluent: water/MeCN*0.1% TFA). The product-containing fractionswere combined and lyophilized to yield 4.4 mg (54%) of the product asthe TFA salt. ¹H NMR (300 MHz, DMSO-d6): δ=1.05 (m, 6H), 1.60 (m, 2H),2.10 (s, 3H), 2.67 (q, J=7.5 Hz, 2H), 2.63 (q, J=6.9 Hz, 2H), 2.93 (m,2H), 3.45 (m, 4H), 5.24 (s, 2H), 5.36 (m, 1H) ppm.; ³¹P NMR (121 MHz,DMSO-d6): δ=16.93 ppm; MS=398 [M⁺+1].

Example 184 Preparation of Representative Compounds of 84

Representative compounds of the invention can be prepared as illustratedbelow.

2-({4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phenoxy-phosphinoylamino)-propionicAcid Ethyl Ester

4-[6′-ethyl-7′-methyl-3′-oxo-4′-(2″-trimethylsilanyl-ethoxy)-1′,3′-dihydro-isobenzofuran-5′-yl]-2-methyl-but-2-en-phosphonicacid (44.8 mg, 0.101 mmol), dicyclohexylcarbodiimide (52.6 mg, 0.254mmol), and phenol (95.8 mg, 1.018 mmol) were dissolved in pyridine (0.3mL) and heated at 70° C. for 4 hrs. The reaction mixture was cooled toroom temperature and the pyridine was removed in vacuo. The crudephosphonic acid diphenyl ester material was partitioned between DCM andHCl (0.1N). The aqueous layer was extracted with DCM and the combinedorganic layers were dried over sodium sulfate. Filtration andevaporation of solvents in vacuo yielded a crude material, which wasused in the next step without further purification.

The crude material was dissolved in MeCN (0.8 mL) and water (0.3 mL).Aqueous sodium hydroxide solution (2N, 0.8 mL) was added in portions(0.2 mL). After all starting material was consumed, the organic solventwas removed in vacuo and the crude material was partitioned betweenchloroform and aqueous HCl (1N). The aqueous layer was extracted withchloroform. The combined organic layers were dried over sodium sulfate.Filtration and evaporation of solvents yielded the crude product as amixture of mono phenyl ester and the symmetrical anhydride.

The crude material of the previous step and ethyl (L)-alaninehydrochloride salt (78.1 mg, 0.509 mmol) were dissolved in DMF (0.4 mL).DMAP (1.2 mg, catalytic) was added, followed by diisopropylethylamine(131.3 mg, 1.018 mmol). Stirring at room temperature was continued.After 20 minutes complete conversion of the anhydride was observed.After 2 hrs PyBOP (101 mg, 0.202 mmol) was added and stirring at roomtemperature was continued. The reaction was filtered and the crudereaction solution was purified by RP-HPLC (eluent: water/MeCN). Theproduct-containing fractions were combined and lyophilized to yield theproduct (15.7 mg, 25% over three steps) as a white powder. ¹H NMR (300MHz, CDCl₃): δ=0.03 (s, 9H), 1.13-1.28 (m, 8H), 2.03 (s, 3H), 2.19 (s,3H), 2.62-2.74 (m, 4H), 3.38 (m, 1H), 3.53 (t, J=6.3 Hz, 2H), 4.03 (m,3H), 4.30 (m, 2H), 5.14 (s, 2H), 5.31 (m, 1H), 7.11-7.17 (m, 3H),7.25-7.30 (m, 2H) ppm; ³¹P NMR (121 MHz, CDCl₃): δ=27.04, 27.73 ppm;MS=615 [M⁺+1].

2-{[4-(6-Ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phenoxy-phosphinoylamino}-propionicAcid Ethyl Ester

2-({4-[6-Ethyl-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phenoxy-phosphinoylamino)-propionicacid ethyl ester (7.5 mg, 0.012 mmol) was dissolved in TFA/DCM (10%, 0.3mL) at −20° C. The reaction mixture was warmed to 0° C. and stirred atthis temperature for 45 minutes. Pyridine (0.09 mL) was added thesolvents were removed in vacuo. The crude material was purified byRP-HPLC (eluent: water/MeCN). The product-containing fractions werecombined and lyophilized, yielding a white powder (5.5 mg, 87%). ¹H NMR(300 MHz, CDCl₃): δ=1.12-1.29 (m, 6H), 2.03 (s, 3H), 2.17 (s, 3H),2.65-2.74 (m, 4H), 3.38 (m, 1H), 3.53 (t, J=6.3 Hz, 2H), 4.03 (m, 3H),5.22 (s, 2H), 5.36 (m, 1H), 7.11-7.16 (m, 3H), 7.24-7.30 (m, 2H), 7.72(m, 1H) ppm; ³¹P NMR (121 MHz, CDCl₃): δ=27.11, 27.57 ppm; MS=515[M⁺+1].

Example 185 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

6-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicAcid

A mixture of6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid methyl ester (1.5 g, 3.45 mmol) and sodium hydroxide (552 mg) in amixture of methanol (20 mL) and water (7 mL) was stirred at roomtemperature for one hour. The solution was acidified with 1N HCl. Theprecipitate was collected by suction filtration and washed with water togive the desired product (1.2 g, 83%). ¹H NMR (300 MHz, CDCl₃) δ 0.02(s, 9H), 1.15-1.22 (m, 2H), 1.76 (s, 3H), 2.13 (s, 3H), 2.12-2.28 (m,2H), 2.35-2.41 (m, 2H), 3.37 (d, 2H, J=7 Hz), 3.71 (s, 3H), 4.22-4.28(m, 2H), 5.07 (s, 2H), 5.13-5.17 (m, 1H) ppm; MS (m/z) 419.3 [M−H]⁻,443.2 [M+Na]⁺.

({6-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoylamino}-methyl)-phosphonicAcid Diethyl Ester

To a solution of6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid (50 mg, 0.12 mmol) in THF (1 mL) was added isobutyl chloroformate(17 μL, 0.13 mmol) and triethylamine (50 μL, 0.36 mmol) at 0° C. Afterstirring at 0° C. for 2 hours, diethyl(aminomethyl)phosphonate oxalate(62 mg, 0.26 mmol) was added and stirring was continued at roomtemperature for 20 minutes. After removal of solvent, the residue waspurified by preparative reverse-phase HPLC to afford 54.8 mg (81%) ofthe desired product. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s, 9H), 1.15-1.22(m, 2H), 1.31 (t, 6H), 1.81 (s, 3H), 2.18 (s, 3H), 2.30 (m, 4H), 3.41(d, 2H, J=7 Hz), 3.65 (dd, 2H, J=6, 12 Hz), 3.77 (s, 3H), 3.77-4.16 (m,4H), 4.26-4.32 (m, 2H), 5.12 (s, 2H), 5.17-5.19 (m, 1H), 5.86 (bs, 1H)ppm; ³¹P (121.4 MHz, CDCl₃) δ 23.01 ppm; MS (m/z) 568 [M−H]⁻, 592[M+Na]⁺.

{[6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoylamino]-methyl}-phosphonicAcid

To a solution of({6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoylamino}-methyl)-phosphonicacid diethyl ester (40 mg, 0.07 mmol) in acetonitrile (1 mL) was addedTMSBr (91 μL, 0.7 mmol) followed by 2,6-lutidine (81.5 μL, 0.7 mmol).The reaction was allowed to proceed overnight when it was completed asjudged by LCMS. The reaction mixture was quenched with MeOH andconcentrated to dryness. The residue was purified by preparativereverse-phase HPLC to afford 2.6 mg (9%) of desired product as a whitesolid. ¹H NMR (300 MHz, CD₃OD) δ 1.67 (s, 3H), 2.17 (m, 5H), 2.30-2.46(m, 2H), 2.80-2.86 (m, 2H), 3.55 (m, 2H), 3.82 (s, 3H), 5.26 (s, 3H)ppm; ³¹P (121.4 MHz, CD₃OD) δ 10.27 ppm; MS (m/z) 412 [M−H]⁻, 414[M+H)⁺.

Example 186 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

(2-{6-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoylamino}-ethyl)-phosphonicAcid Diethyl Ester

To a solution of6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid (50 mg, 0.12 mmol) in THF (1 mL) was added isobutyl chloroformate(17 μL, 0.13 mmol) and triethylamine (50 μL, 0.36 mmol) at 0° C. Afterstirring at 0° C. for 2 hours, diethyl (aminoethyl) phosphonate oxalate(62 mg, 0.26 mmol) was added and stirred at room temperature wascontinued for one hour. After removal of solvent, the residue waspurified by preparative reverse-phase HPLC to afford 37 mg (54%) of thedesired product as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s,9H), 1.15-1.22 (m, 2H), 1.31 (t, 6H), 1.81 (s, 3H), 1.85-1.93 (m, 2H),2.18 (s, 3H), 2.30 (m, 4H), 3.41 (d, 2H, J=7 Hz), 3.48-3.54 (m, 2H),3.77 (s, 3H), 3.77-4.16 (m, 4H), 4.26-4.32 (m, 2H), 5.12 (s, 2H),5.17-5.19 (m, 1H), 6.30 (bs, 1H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 29.91ppm; MS (m/z) 584 [M+H]⁺.

{2-[6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-hex-4-enoylamino]-ethyl}-phosphonicAcid

To a solution of(2-{6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoylamino}-ethyl)-phosphonicacid diethyl ester (36.6 mg, 0.063 mmol) in acetonitrile (1 mL) wasadded TMSBr (81 μL, 0.63 mmol) followed by 2,6-lutidine (73 μL, 0.63mmol). The reaction was allowed to proceed overnight when it wascompleted as judged by LCMS. The reaction mixture was quenched with MeOHand concentrated to dryness. The residue was purified by preparativereverse-phase HPLC to afford 5.8 mg (29%) of desired product as a whitesolid. ¹H NMR (300 MHz, CD₃OD) δ 1.80 (s, 3H), 2.14 (m, 5H), 2.25 (m,4H), 3.35 (m, 2H), 3.38-3.38 (m, 2H), 3.75 (s, 3H), 5.23 (s, 3H) ppm;³¹P (121.4 MHz, CD₃OD) δ 26.03 ppm; MS (m/z) 426 [M−H]⁻, 428 [M+H]⁺.

Example 187 Preparation of Representative Compounds of the Invention

Representative compounds of the invention can be prepared as illustratedbelow.

{4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicAcid Diphenyl Ester

To a solution of[{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicacid (260 mg, 0.59 mmol) in DMF (6 mL) and phenol (555 mg, 5.9 mmol) wasadded dicyclohexyl carbodiimide (1.21 g, 5.9 mmol) and DMAP (36 mg,0.295 mmol). The reaction mixture was heated to 140° C. for 30 mins.After cooling to room temperature the mixture was partitioned betweenEtOAc/Hexane (1:1) and 5% aqueous LiCl solution. The organic layer waswashed with 5% aqueous LiCl solution repeatedly, then dried over Na₂SO₄.After removal of solvent, the residue was purified by silica gelchromatography to provide 75 mg (21%) of the desired product. MS (m/z)617 [M+Na]⁺.

{4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicAcid Monophenyl Ester

To a solution of{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicacid diphenyl ester (75 mg, 0.126 mmol) in THF (5 mL) was added 1N NaOH(0.1 mL) solution. The mixture was allowed to stir at room temperaturefor 16 hours. EtOAc was added and the resulting mixture was washed with1H HCl. The organic layer was concentrated to dryness and the residuewas purified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 24.8 mg (38%) of the desiredproduct. MS (m/z) 517 [M−H]⁻, 541 [M+Na]⁺.

2-({4-[6-Methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phenoxy-phosphinoyloxy)-propionicAcid Ethyl Ester

To a solution of{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicacid monophenyl ester (25 mg, 0.048 mmol) and ethyl (S)-(−)-lactate (34mg, 0.288 mmol) in pyridine (1 mL) was added PyBOP (125 mg, 0.24 mmol).The solution was stirred at room temperature for 16 hours andconcentrated. The residue was purified by RP HPLC using a C18 columnwith a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 24 mg(83%) of the desired product. MS (m/z) 641 [M+Na]⁺.

2-{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phenoxy-phosphinoyloxy}-propionicAcid Ethyl Ester

To a solution of2-({4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phenoxy-phosphinoyloxy)-propionicacid ethyl ester (24 mg, 0.039 mmol) in DCM (1 mL) was added TFA (0.5mL) and the mixture was stirred at room temperature for 10 minutes. Thereaction mixture was dried under reduced pressure and the residue waspurified by RP-HPLC to provide 18 mg (90%) of the desired product as aclear oil. ¹H NMR (300 MHz, CDCl₃) δ 1.18-1.34 (m, 3H), 1.36-1.48 (dd,3H), 2.02 (m, 3H), 2.17 (s, 3H), 2.78-2.98 (dd, 2H), 3.45 (m, 2H), 3.79(s, 3H), 4.05-4.25 (m, 2H), 4.97 (m, 1H), 5.21 (s, 2H), 5.48 (t, J=7.2Hz, 1H), 7.05-7.18 (m, 5H) ppm; ³¹P (121.4 MHz, CDCl₃) δ 24.59, 26.13ppm; MS (m/z) 517 [M−H]⁻, 519 [M+H]⁺.

Example 188 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

2-{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phenoxy-phosphinoyloxy}-propionicAcid

To a solution of2-{[4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phenoxy-phosphinoyloxy}-propionicacid ethyl ester (10 mg, 0.019 mmol) in THF (3 mL) was added 1N NaOH(232 μL) and the mixture was stirred at room temperature for 1 hour. Thereaction mixture was dried under reduced pressure and the residue waspurified by RP-HPLC to provide 6 mg (77%) of the desired product as aclear oil. ¹H NMR (300 MHz, CD₃OD) δ 1.41 (d, J=7 Hz, 3H), 1.97 (s, 3H),2.16 (s, 3H), 2.59 (d, J=22 Hz, 2H), 3.45 (m, 2H), 3.79 (s, 3H), 4.83(m, 1H), 5.26 (s, 2H), 5.43 (t, J=7.2 Hz, 1H) ppm; ³¹P (121.4 MHz,CD₃OD) δ 27.02 ppm; MS (m/z) 413 [M−H]⁻, 415 [M+H]⁺.

Example 189 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

2-{[4-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyl]-phenoxy-phosphinoylamino}-propionicAcid Ethyl Ester

{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}-phosphonicacid monophenyl ester (1 g, ˜1.9 mmol) was combined with pyBOP (2 g, 4mmol) and DMAP (120 mg, 0.96 mmol). A solution of L-alanine ethyl esterhydrochloride salt (2.9 g, 19 mmol) and diisopropylethylamine (6.7 mL,38 mmol) in pyridine (5 mL) was added to the monoacid mixture and thereaction was stirred at room temperature for 12 hours. The reactionmixture was then concentrated and purified twice by columnchromatography (1% MeOH/CH₂Cl₂ 3% MeOH/CH₂Cl₂). The resulting oil wasdissolved in a vigorously-stirred solution of 10% TFA/CH₂Cl₂ (30 mL) at−40° C. The reaction was gradually warmed to 0° C. After about 3 hours,the reaction was complete. Pyridine (4.5 mL) was added, and the reactionmixture was concentrated. The product was purified by preparative TLC(5% MeOH/CH₂Cl₂) and concentrated to give 210 mg (21%) of the desiredproduct as a light yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.83-7.70 (m,1H), 7.30-7.20 (m, 2H), 7.18-7.03 (m, 3H), 5.60-5.35 (m, 1H), 5.21 (s,2H), 4.17-3.95 (m, 3H), 3.79 (s, 3H), 3.60-3.40 (m, 3H), 2.80-2.60 (m,2H), 2.17 (m, 3H), 2.01 (m, 3H), 1.30-1.10 (m, 6H) ppm; ³¹P NMR (121MHz, CDCl₃) δ 28.0, 27.5 ppm; MS (m/z) 516 [M−H]⁻.

Example 190 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

2-(Dimethoxy-phosphoryl)-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicAcid Methyl Ester

To a solution of trimethylphosphonoacetate (63 μL, 0.39 mmol) in THF (1mL) was added NaN(TMS)₂ (0.39 mmol, 0.39 mL) at ambient temperature.After 30 minutes, a solution of6-(4-bromo-3-methyl-but-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(69 mg, 0.156 mmol) in THF (1 mL) was added. The reaction mixture wasstirred for 2 hours when a precipitate was observed. The reactionmixture was worked up by addition of a saturated aqueous solution ofammonium chloride and extraction of the product with EtOAc. The organicextract was dried and the product was purified using silica gelchromatography with 0-100% EtOAc-Hexanes to provide 40 mg of the desiredproduct as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 0.05 (s, 9H),1.20-1.26 (m, 2H), 1.79 (s, 3H), 2.17 (s, 3H), 2.42-2.72 (m, 2H), 3.19(ddd, 1H, J=4, 12, 23 Hz), 3.39 (d, 2H, J=7 Hz), 3.62 (s, 3H), 3.75 (s,3H), 3.77-3.84 (m, 6H), 4.27-4.34 (m, 2H), 5.12 (s, 2H), 5.24 (t, 1H,J=7 Hz) ppm; ³¹P (121.4 MHz, CDCl₃) δ 25.1 ppm; MS (m/z) 565.2 [M+Na]⁺.

6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-4-methyl-2-phosphono-hex-4-enoicAcid Methyl Ester

To a solution of2-(dimethoxy-phosphoryl)-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid methyl ester (30 mg, 0.055 mmol) in acetonitrile (2 mL) was addedtrimethylsilyl bromide (0.18 mL). After 10 minutes, 2,6-lutidine (0.16mL) was added to the reaction at ambient temperature. The reaction wasallowed to proceed for 16 hours before it was concentrated to dryness.The residue was resuspended in a solution of DMF: H₂O (8:2, 1 mL) andpurified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 18 mg of the product as a whitepowder. ¹H NMR (300 MHz, CD₃OD) δ 1.81 (s, 3H), 2.16 (s, 3H), 2.40-2.49(m, 1H), 2.63 (dt, 1H, J=6, 17 Hz), 3.07 (ddd, 1H, J=4, 12, 23 Hz), 3.38(3, 2H, J=7 Hz), 3.52 (s, 3H), 3.77 (s, 3H), 5.25 (s, 2H), 5.28 (t, 1H,J=7 Hz) ppm; ³¹P (121.4 MHz, CDCl₃) δ 19.5 ppm; MS (m/z) 415.2 [M+H]⁺,437.2 [M+Na]⁺.

Example 191 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

2-(Bis-(2,2,2-trifluoroethoxy)phosphoryl)-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoic Acid Methyl Ester

To a solution of [bis-(2,2,2-trifluoro-ethoxy)-phosphoryl]-acetic acidmethyl ester (186 μL, 0.88 mmol) in anhydrous THF (2 mL) was added asolution of 1N NaN(TMS)₂ in THF (0.88 mL, 0.88 mmol). The solution wasstirred at room temperature for 30 minutes, whereupon a solution of6-(4-bromo-3-methyl-but-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(98 mg, 0.22 mmol) in THF (1 mL) was added. The reaction mixture wasstirred overnight when a precipitate was observed. The reaction mixturewas worked up by addition of a saturated aqueous solution of ammoniumchloride and extraction of the product with EtOAc. The organic extractwas dried and the product was purified by RP HPLC using a C18 columnwith a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA to provide 72 mg(48%) of the product as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 0.05(s, 9H), 1.22 (t, 3H, J=7 Hz), 1.81 (s, 3H), 2.18 (s, 3H), 2.5-2.7 (m,2H), 3.3 (ddd, 1H, J=4, 12, 23 Hz), 3.40 (d, 2H, J=7 Hz), 3.65 (s, 3H),3.76 (s, 3H), 4.29-5.13 (m, 6H), 5.13 (s, 2H), 5.28 (t, 1H, J=7 Hz) ppm;MS (m/z) 701.2 [M+Na]⁺.

2-(Bis-(2,2,2-trifluoroethoxy)phosphoryl)-6-[6-methoxy-7-methyl-3-oxo-4-(2-hydroxyoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicAcid Methyl Ester

[2-(Bis-(2,2,2-trifluoroethoxy)phosphoryl)-6-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-4-methyl-hex-4-enoicacid methyl ester (70 mg) was dissolved in a solution of 10%trifluoroacetic acid in dichloromethane (5 mL). After 10 minutes themixture was concentrated and the product was purified by RP HPLC using aC18 column with a gradient of H₂O, 0.1% TFA-acetonitrile, 0.1% TFA toprovide 45 mg (75%) of the product as a colorless oil. ¹H NMR (300 MHz,CDCl₃) δ 1.81 (s, 3H), 2.16 (s, 3H), 2.5-2.7 (m, 2H), 3.3 (ddd, 1H),3.38 (d, 2H, J=7 Hz), 3.65 (s, 3H), 3.77 (s, 3H), 4.33-4.43 (m, 4H),5.21 (s, 2H), 5.33 (t, 1H, J=7 Hz) ppm; ³¹P (121.4 MHz, CDCl₃) δ 25.8ppm; MS (m/z) 601.2 [M+Na]⁺.

Example 192 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

6-(4-Hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-[hydroxy-(2,2,2-trifluoro-ethoxy)-phosphoryl]-4-methyl-hex-4-enoicAcid

To a solution of the bis-(2,2,2-trifluoro-ethoxy)-phosphoryl methylester (186 μL, 0.88 mmol) in anhydrous THF (0.5 mL) was added a solutionof 1N NaOH (aqueous; 0.06 mL) and N-methylpyrrolidinone (0.2 mL). After6.5 hours, another aliquot of 1N NaOH (0.06 mL) was added and themixture was stirred overnight. After concentration, the residue wassuspended in DMF (<1 mL), neutralized with a few drops of TFA andpurified by RP HPLC using a C18 column with a gradient of H₂O, 0.1%TFA-acetonitrile, 0.1% TFA to provide 5.6 mg (72%) of the product as awhite powder after lyophilization. ¹H NMR (300 MHz, CD₃OD) δ 1.83 (s,3H), 2.16 (s, 3H), 2.43-2.51 (m, 1H), 2.59-2.70 (m, 1H), 3.13 (ddd, 1H),3.40 (d, 2H), 3.76 (s, 3H), 4.36-4.47 (m, 2H), 5.25 (s, 2H), 5.34 (t,1H, J=7 Hz) ppm; MS (m/z) 505.2 [M+Na]⁺.

Example 193 Preparation of Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedbelow.

Phosphorous Acidmono-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}ester

To a solution of6-(4-hydroxy-3-methyl-but-2-enyl)-5-methoxy-4-methyl-7-(2-trimethylsilanyl-ethoxy)-3H-isobenzofuran-1-one(75 mg, 0.20 mmol) and DIEA (49 μL, 0.28 mmol) in dioxane (2 mL) wasadded 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (56.7 mg, 0.28 mmol)according the procedure of Shadid, B. et al., Tetrahedron, 1989, 45, 12,3889. After 10 minutes, another portion of2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (40 mg, 0.20 mmol) and DIEA(35 μL, 0.20 mmol) were added. The reaction was allowed to proceed atroom temperature for an additional hour, after which it was quenched bythe addition of H₂O. The solution was stirred for another 10 minutes andconcentrated in vacuo to a small volume. The product was triturated withdiethyl ether and coevaporated from acetonitrile (4×10 mL) to providethe product. ¹H NMR (300 MHz, CDCl₃) δ 0.03 (s, 9H), 1.08-1.30 (m, 2H),1.84 (br s, 3H), 2.17 (s, 3H), 3.46 (br s, 2H), 3.76 (s, 3H), 4.21-4.39(m, 4H), 5.12 (s, 2H), 5.43-5.60 (m, 1H), 7.83 (br s, 1H); ³¹P (121.4MHz, CDCl₃) δ 7.22; MS (m/z) 441 [M−H]⁻.

Example 194 Preparation of Representative Compounds of 81

Representative compounds of the invention can be prepared as illustratedbelow.

Phosphoric acidmono-{4-[6-methoxy-7-methyl-3-oxo-4-hydroxy-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}ester

A solution of phosphorous acidmono-{4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enyl}ester(27 mg, 0.06 mmol) in dioxane (1 mL) was stirred with DIEA (21 μL, 0.12mmol) and N,O-bis(trimethylsilyl)acetamide (29 μL, 0.12 mmol) at roomtemperature for 3 hours. To the reaction solution was added2,2′-dipyridyldisulfide (16 mg, 0.072 mmol) and the mixture was allowedto stir for an additional 2 hours at room temperature. The reactionmixture was diluted by addition of H₂O and the solution was stirred for2 more hours when it was concentrated. The residue was dissolved in asolution of 10% TFA/CH₂Cl₂ and stirred at room temperature for 9 hours.The reaction mixture was dried under reduced pressure and the productwas purified by reverse-phase HPLC to provide the desired product as awhite solid. ¹H NMR (300 MHz, CD₃OD) δ 1.87 (s, 3H), 2.16 (s, 3H), 3.47(d, 2H, J=7 Hz), 3.79 (s, 3H), 4.28 (d, 2H, J=6 Hz), 5.26 (s, 2H),5.50-5.61 (m, 1H); ³¹P (121.4 MHz, CD₃OD) δ 0.50; MS (m/z) 357 [M−H]⁻.

Examples 195-199

Synthetic methodologies and intermediate compounds that can be used toprepare analogs of formulae H, I, J, and K are described in Examples195-199. These compounds are representative examples of compounds ofFormulae 77-80.

Three regions of mycophenolate mofetil can be utilized for theattachment of the phosphonate prodrug onto mycophenolic acid asdemonstrated by compounds H, I, and K shown above. Also, the carboxylicacid can be replaced with a phosphonic acid that is part of the prodrugmoiety as in compound J.

Example 195 Specific Embodiments of the Invention of Formula 81

Representative compounds of the invention are illustrated above.

Example 196 General Route to Representative Compounds of Formula 81

Representative compounds of the invention can be prepared as illustratedabove. The morpholino ethyl moiety can serve as a prodrug functionalityto improve bioavailability and can be replaced with the phosphonateprodrug handle as shown above. Mycophenolic acid is commerciallyavailable, e.g., from Sigma Chemical Company, St. Louis, Mo. Activationof the carboxylic acid 196.1 in the presence of the free phenol,followed by addition of an alcohol carrying the phosphonate group,results in the formation of the desired product 196.3 (U.S. Pat. No.4,786,637).

Specifically, mycophenolic acid 196.1 is dissolved in dichloromethane.Thionyl chloride is added followed by a catalytic amount of DMF. Thereaction mixture is stirred at room temperature for 3 hours, after whichthe volatile components are removed under vacuum. Thephosphonate-alcohol is dissolved in dichloromethane and chilled to about4° C. on an ice bath. The mycophenolic acid chloride 196.2 is dissolvedin dichloromethane and added to the chilled solution. After stirring for90 minutes at about 4° C., the reaction mixture is washed with water andthen with aqueous sodium bicarbonate. The organic solution is dried andevaporated to yield the phosphonate prodrug 196.4.

Example 197 Synthesis of Representative Compounds of Formula 79

Representative compounds of the invention can be prepared as illustratedabove. The C-4 phenol position provides a reactive handle for furtheranalogs as illustrated above. Once the carboxylic acid of 197.1 isblocked by morpholino ethyl, such as in compound 197.2, or a phosphonateprodrug as in compound 197.3, the phenol can be alkylated under basicconditions. Bases such as pyridine, potassium carbonate, ortriethylamine are utilized. Leaving groups such astrifluoromethylsulfonate, mesylate, bromide, or iodide are attached tothe phosphonate prodrug subunit and reacted, in the presence of base,with compound 197.2. Compound 197.3 can either be used directly, or inthe form of a salt, compound 197.4. Among the large number of salts thatcan be prepared, chloride and bisulfate salts are of particular utility.

Preparation of compound 197.4 is outlined in more detail above. Compound197.2 is prepared similar to compound 196.2 (described in an Exampleabove). A solution of morpholino ethanol in dichloromethane is cooled toabout 4° C. The mycophenolic acid chloride 197.1.1 is dissolved indichloromethane and added to the cooled solution. Stirring this solutionfor about 90 minutes gives compound 197.2. The reaction mixture iswashed with water and dried with sodium sulfate. Removal of the solventprovides isolated compound 197.2. Alkylation at the phenolic position of197.2 is achieved by suspending the compound in pyridine. Triflate197.2.1 is added to the solution and the mixture is stirred at roomtemperature for about 90 minutes. The reaction mixture is poured intowater and the product is extracted with ethyl acetate. Removal of theorganic layer provides compound 197.3. Hydrochloride salt of 197.3 canoptionally be prepared. Compound 197.3 is dissolved in isopropanol andthe solution is added to a mixture of hydrogen chloride in isopropanol.The hydrochloride salt 197.4 is collected by filtration and dried undervacuum.

Example 198 Synthesis of Representative Compounds of Formula 78

Representative compounds of the invention can be prepared as illustratedabove. The carboxylic acid of mycophenolic acid can be replaced with aphosphonic acid that may also serves as a prodrug handle. In order toremove the carboxylic acid containing side chain, the acid chloride197.2 (prepared in Example 197) is converted to ester 198.1. Protectionof the phenol with a silyl group, followed by dihydroxylation andcleavage of the diol generates aldehyde 198.3 (Pankiewicz, et al., J.Med. Chem. 2002, 45, 703), (Patterson et al., U.S. Pat. No. 5,444,072).A Wittig reaction with ylide 198.3.1 carrying an appropriately protectedphosphonate provides the desired compound 198.4. Final deprotectionyields compound 198.5.

Mycophenolate ester 198.1 can simply be prepared by stirring the acidchloride 198.1.1 with MeOH. Then, the phenol position of mycophenolateester is protected by a silyl group such as TBS to provide compound198.2. Once the phenol position is protected, dihydroxylation usingosmium tetraoxide followed by periodinate cleavage provides aldehyde198.3. Aldehyde 198.3 and excess of the ylide 198.3.1 are heated inbenzene at reflux for about 24 hours. The reaction mixture isconcentrated and the residue is purified by column chromatography toprovide olefin 198.4 (Pankiewics et al., J. Med. Chem. 2002, 45, 703). Afinal deprotection using HF-pyridine yields the final product 198.5.

Example 199 Synthesis of Representative Compounds of Formula 80

Representative compounds of the invention can be prepared as illustratedabove. A phosphonate-attachment point can be unmasked afterdemethylation of mycophenolate ester 199.1.1. For this purpose, the 4-OHneeds to be masked with a protecting group such as a silyl group. Oncethe 6-OMe is demethylated and alkylated, the protecting group atposition 4 is removed to reveal the final product 199.4. The morphonylethanol group is installed early and carried through the alkylationsteps. A different protecting group may be installed initially andremoved later. In such a synthesis, the last step is the formation ofthe morpholinoethyl ester compound.

Synthesis of compound 199.4 is shown above. Phenol 199.1.1 is protectedwith TBS group in CH₂Cl₂ using imidazole as base to yield 199.5.Demethylation is performed using thiolate nucleophiles to generatecompound 199.6. A variety of other methods are also available inliterature as described in Protective Groups in Organic Synthesis byGreene and Wuts. Alklation of the 6-OH using a triflate of thephosphonate prodrug proceeds well using K₂CO₃ or TEA to provide 199.7.Final deprotection to remove the TBS group provides product 199.4.

Example 200 Preparation of Representative Compounds of the Invention

Representative compounds of the invention can be prepared as illustratedabove and in the following schemes.

Synthesis of Phenacetaldehydes with Variants at R₁, R₂

The parent compound (R₁=OMe; R₂=Me) is accessible by semi-synthesis frommycophenolic acid as follows:

To a solution of mycophenolic acid (500 g, 1.56 mol) in MeOH (4 L) undernitrogen atmosphere was added sulfuric acid (10 mL) dropwise, and thesuspension was stirred at room temperature. After 2 hours the reactionbecame homogeneous, and soon thereafter a precipitate was formed. Thereaction was allowed to stir at room temperature for 10 hours at whichtime TLC indicated complete reaction. The reaction was cooled in an icebath to 10° C. and then filtered using a Buchner funnel. The filter cakewas washed with ice cold methanol (750 mL) followed by hexanes (750 mL)and then dried to give 497 g (95%) of the desired product 200.3 as asolid: ¹H NMR (300 MHz, CDCl₃) δ, 1.81 (s, 3H), 2.18 (s, 3H), 2.15 (s,3H), 2.37-2.50 (m, 4H), 3.38 (d, 2H, J=7 Hz), 3.62 (s, 3H), 3.77 (s,3H), 5.13 (s, 2H), 5.22 (m, 1H), 7.17 (s, 1H).

To a solution of 200.4 (3.99 g, 11.9 mmol), PPh₃ (4.68 g, 17.9 mmol),and diisopropyl azodicarboxylate (3.46 mL, 17.9 mmol) in THF (60 mL) at0° C. was added a solution of 2-trimethylsilylethanol (2.05 mL, 14.3mmol) in THF (20 mL). The resulting yellow solution was allowed to warmto room temperature and stirred for 4 hours. The reaction was worked upby concentrating the solution to dryness and addition of ether andhexanes. Triphenylphosphine oxide was removed by filtration and thefiltrate was concentrated and purified by silica gel chromatography toprovide 4.8 g (100%) of 200.5 as a clear oil: ¹H NMR (300 MHz, CDCl₃) δ0.03 (s, 9H), 1.18-1.30 (m, 2H), 1.81 (s, 3H), 2.18 (s, 3H), 2.25-2.33(m, 2H), 2.37-2.45 (m, 2H), 3.42 (d, 2H, J=7 Hz), 3.62 (s, 3H), 3.77 (s,3H), 4.25-4.35 (m, 2H), 5.13 (s, 2H), 5.12-5.22 (m, 1H).

A solution of 200.5 (9.6 g, 22 mmol) in MeOH (90 mL), CH₂Cl₂ (90 mL) andpyridine (0.7 mL) was cooled to −70° C. using a dry ice/acetone bath. Astream of ozone was bubbled through the reaction via a gas dispersiontube until the reaction became blue in color (1.5 hours). The ozone linewas replaced with a stream of nitrogen and bubbling continued foranother 30 minutes, by which time the blue color had disappeared. Tothis solution at −70° C. was added thiourea (1.2 g, 15.4 mmol) in oneportion, and the cooling bath was removed. The reaction was allowed towarm to room temperature and stirred for 15 hours. The reaction wasworked up by filtration to remove solid thiourea S-dioxide, and thenpartitioned between CH₂Cl₂ and water. The organic layer was removed. Theaqueous layer was washed with CH₂Cl₂ and the organic extracts werecombined, washed with aqueous 1N HCl, saturated NaHCO₃ and brine, anddried in vacuo. The residue was purified by silica gel chromatography toafford 7.3 g (99%) of 200.6 as a white solid: ¹H NMR (300 MHz, CDCl₃) δ−0.01 (s, 9H), 1.05-1.15 (m, 2H), 2.15 (s, 3H), 3.69 (s, 3H), 3.78 (d,2H, J=1 Hz), 4.27-4.39 (m, 2H), 5.11 (s, 2H), 9.72 (d, 1H, J=1 Hz).

Example 201 Preparation of Representative Compounds of the Invention R₁Variants of Example 200:

Representative compounds of the invention can be prepared as illustratedabove. The starting material, synthesized according to J. Med. Chem.,1996, 39, 4181-4196, is transformed to the desired aldehyde usingmethods analogous to those described above.

The starting material, synthesized according to J. Med. Chem., 1996, 39,4181-4196, is transformed to the desired aldehyde using methodsanalogous to those described above.

The starting material, synthesized according to J. Med. Chem., 1996, 39,4181-4196, is transformed to the desired aldehyde using methodsanalogous to those described above.

The aldehyde is dissolved in an organic solvent such as methanol andsodium borohydride is added. At the end of the reaction, aqueous HClsolution is added and the solvent is removed in vacuo. Furtherpurification is achieved by chromatography.

The resulting alcohol is dissolved in an organic solvent such asdichloromethane (DCM). Pyridine and acetic anhydride are added andstirring at room temperature is continued. At the end of the reactionadditional DCM is added and the solution is washed with aqueous HClsolution, aqueous sodium bicarbonate solution, and dried over sodiumsulfate. Filtration and evaporation of the solvent in vacuo gives thecrude product. Further purification is achieved by chromatography.

The acetate is dissolved in DCM and bromine is added, according to aprocedure from J. Med. Chem., 1996, 39, 4181-4196. At the end of thereaction additional DCM is added and the solution is washed with aqueoussodium thiosulfate solution and brine. The organic layer is dried oversodium sulfate. Filtration and evaporation of solvents yields the crudematerial. Further purification is achieved by chromatography.

The product of the previous step, lithium chloride, triphenylarsine,tributylvinyltin, andtris(dibenzylideneacetone)dipalladium(0)-chloroform adduct are heated inan organic solvent such as N-methylpyrrolidinone at an elevatedtemperature of approximately 55° C., according to a procedure from J.Med. Chem., 1996, 39, 4181-4196. At the end of the reaction the mixtureis cooled to room temperature and poured into a mixture of ice,potassium fluoride, water, and ethyl acetate. Stirring is continued forone hour. The suspension is filtered through Celite and extracted withethyl acetate. The combined organic extracts are dried over sodiumsulfate. The solvents are removed in vacuo and the crude material isfurther purified by chromatography.

The product of the previous step is dissolved in an organic solvent suchas DCM or THF.1,1,1-tris(acyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (Dess-Martinreagent) is added and the solution is stirred at room temperature,according to a procedure from J. Org. Chem., 1984, 48, 4155-4156. At theend of the reaction diethyl ether is added, followed by aqueous sodiumhydroxide solution. The layers are separated and the organic layer iswashed with aqueous sodium hydroxide solution, water, and dried oversodium sulfate. Filtration and evaporation of solvents yields the crudevinyl product. Further purification is achieved by chromatography.

The starting material is dissolved in an organic solvent such astoluene. P(isobutylNCH₂CH₂)₃N, palladium(II) acetate, sodiumtert-butoxide, and benzylamine are added and the mixture is heated at80° C., according to a procedure from J. Org. Chem., 2003, 68, 452-459.At the end of the reaction the mixture is cooled to room temperature andthe solvents are removed in vacuo. The crude material is purified bychromatography. Any residual acetate is removed by brief treatment withmethanolic sodium methoxide.

The benzyl-protected aniline is dissolved in an organic solvent such asDMF. Palladium on carbon is added and the reaction mixture is placedunder an atmosphere of hydrogen. At the end of the reaction the mixtureis filtered through Celite. The solvents are removed in vacuo. Furtherpurification is achieved by chromatography.

The resulting primary aniline is dissolved in an organic solvent such asTHF, acetonitrile, or DMF and is treated with formaldehyde and sodiumtriacetoxyborohydride as described in J. Org. Chem., 1996, 61,3849-3862. The reaction is quenched with aqueous sodium bicarbonate andthe product is extracted with an organic solvent such as ethyl acetate.The crude material is treated with di-t-butyl dicarbonate in an organicsolvent such as dimethylformamide and aqueous sodium hydroxide. Theresulting carbamate is purified by chromatography.

The primary alcohol product is dissolved in an organic solvent such asDCM or THF. 1,1,1-tris(acyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one(Dess-Martin reagent) is added and the solution is stirred at roomtemperature, according to a procedure from J. Org. Chem., 1984, 48,4155-4156. At the end of the reaction diethyl ether is added, followedby aqueous sodium hydroxide solution. The layers are separated and theorganic layer is washed with aqueous sodium hydroxide solution, water,and dried over sodium sulfate. Filtration and evaporation of solventsyields the crude aldehyde product. Further purification is achieved bychromatography.

The starting material is dissolved in an organic solvent such as DCM orTHF and is treated with the mixed anhydride of formic and pivalic acids,according to a procedure from Recl. Trav. Chem. Pay-Bas, 1982, 101, 460.At the end of the reaction, the solvent and all volatiles are removed invacuo and the crude product is further purified by chromatography.

The product is dissolved in an organic solvent such as DCM or THF.1,1,1-Tris(acyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (Dess-Martinreagent) is added and the solution was stirred at room temperature,according to a procedure from J. Org. Chem., 1984, 48, 4155-4156. At theend of the reaction diethyl ether is added, followed by aqueous sodiumhydroxide solution. The layers are separated and the organic layer iswashed with aqueous sodium hydroxide solution, water, and dried oversodium sulfate. Filtration and evaporation of solvents yields the crudeproduct. Further purification is achieved by chromatography.

Example 202 Preparation of Representative Compounds of the Invention R₂Variants of Examples 200 and 201:

Representative compounds of the invention can be prepared as illustratedabove. The starting material is dissolved in an organic solvent such asDMF and reacted with N-chlorosuccinimide, according to a procedure fromJ. Med. Chem., 1996, 39, 4181-4196. After the starting material isconsumed the reaction mixture is poured into water and the product isextracted with diethyl ether. The combined organic layers are dried oversodium sulfate. Filtration and evaporation of the solvent yields a crudereaction product.

The product of step one is dissolved in a mixture of organic solventssuch as methanol, DCM, and pyridine. The solution is cooled to −78° C.and ozone is bubbled into the solution until a blue color persists. Theexcess ozone is removed with a nitrogen stream. The reaction mixture iswarmed to room temperature and thiourea is added. Stirring at roomtemperature is continued. The reaction mixture is filtered andpartitioned between DCM and water. The aqueous layer is extracted withDCM and the combined organic layers are washed with HCl (1 N), saturatedaqueous sodium bicarbonate solution and brine. The solution is driedover sodium sulfate. Filtration and evaporation of the solvents yieldsthe crude aldehyde. Further purification is achieved by chromatography.

The starting material is dissolved in a mixture of organic solvents suchas methanol, DCM, and pyridine. The solution is cooled to −78° C. andozone is bubbled into the solution until a blue color persists. Theexcess ozone is removed with a nitrogen stream. The reaction mixture iswarmed to room temperature and thiourea is added. Stirring at roomtemperature is continued. The reaction mixture is filtered andpartitioned between DCM and water. The aqueous layer is extracted withDCM and the combined organic layers are washed with HCl (1 N), saturatedaqueous sodium bicarbonate solution, and brine. The solution is driedover sodium sulfate. Filtration and evaporation of the solvents yieldsthe crude aldehyde. Further purification is achieved by chromatography.

The product of step one is dissolved in an organic solvent such asbenzene. Trifluoromethanesulfonyl chloride anddichlorotris(triphenylphosphine)rhuthenium are added and the solution isdegassed. The reaction mixture is heated at 120° C., according to aprocedure from J. Chem. Soc., Perkin Trans. 1, 1994, 1339-1346. At theend of the reaction the mixture is cooled to room temperature and thesolvent is removed in vacuo. Further purification of the trifluoromethylaldehyde product is achieved by chromatography.

Example 203 Preparation of Representative Compounds of Formula 81Synthesis of Olefins and Linkers to Phosphonates

Representative compounds of the invention can be prepared as illustratedin the following schemes.

The phenacetaldehyde (5.3 g, 15.8 mmol) in toluene (50 mL) was heated at100° C. with 2-(triphenyl-phosphanylidene)-propionaldehyde (6.8 g, 20.5mmol) overnight. After concentration, the residue was purified by silicagel chromatography to provide 4.24 g (72%) of the unsaturated aldehydeas a pale yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 0.00 (s, 9H),1.10-1.21 (m, 2H), 1.87 (s, 3H), 2.16 (s, 3H), 3.67-3.76 (m, 2H), 3.74(s, 3H), 4.27-4.39 (m, 2H), 5.11 (s, 2H), 6.40-6.48 (m, 1H), 9.2 (s,1H).

The trimethylsilyethyl protected aldehyde is treated withdiethylphosphite in a solvent such as acetonitrile in the presence of abase such as triethylamine to afford the hydroxy phosphonate, accordingto a procedure such as that reported in Tetrahedron, 1995, 51, 2099. Thehydroxy phosphonate is O-alkylated and then the protecting group isremoved by treatment with either trifluoroacetic acid ortetrabutylammonium fluoride to generate the desired methoxy phosphonateanalog.

Alternatively, the aldehyde is mixed with diethyl(2-aminoethyl)phosphonate and treated with a reducing agent such assodium triacetoxyborohydride to generate the amino phosphonate analog.

A solution of4-[6-methoxy-7-methyl-3-oxo-4-(2-trimethylsilanyl-ethoxy)-1,3-dihydro-isobenzofuran-5-yl]-2-methyl-but-2-enal(103 mg, 0.27 mmol) in methanol (5 mL) was cooled to 0° C. A solution ofCeCl₃ (0.68 mL, MeOH: H₂O, 9:1) was added, followed by LiBH₄ (0.14 mL,0.28 mmol of a 2M solution in THF). The ice bath was removed and thereaction mixture was allowed to warm to room temperature. The reactionmixture was stirred for an additional 40 minutes whereupon TLC indicatedcomplete consumption of starting aldehyde. The reaction was worked up byaddition of aqueous 1N HCl (0.5 mL) and the product was extracted withCH₂Cl₂. The organic layer was washed with saturated aqueous sodiumbicarbonate solution and brine. The organic layer was concentrated underreduced pressure and the residue was purified by silica gelchromatography to provide 100 mg (97%) of the product as a clear liquid.¹H NMR (300 MHz, CDCl₃) δ 0.00 (s, 9H), 1.20 (dd, 2H, J=7, 8 Hz), 1.81(s, 3H), 2.13 (s, 3H), 3.38-3.50 (m, 2H), 3.74 (s, 3H), 3.95 (s, 2H),4.27 (dd, 2H, J=7, 8 Hz), 5.08 (s, 2H), 5.17-5.44 (m, 1H).

Polymer-supported triphenylphosphine is soaked in DCM for 1 hour. Theallylic alcohol and carbon tetrabromide are sequentially added. When thereaction is complete, the mixture is filtered and the filtrateconcentrated. The bromide is purified as necessary by chromatography.

The allylic bromide is treated in an inert organic solvent such asdimethylformamide with an alkali metal salt of ethyldiethoxyphosphorylacetate (prepared by reacting ethyldiethoxyphosphorylacetate with sodium hexamethyldisilazide or sodiumhydride) to afford the ethoxycarbonyl phosphonate, according to aprocedure such as that described in WO 95/22538. The carboxylic estergroup is converted to both the carboxylic amide and the hydroxymethylgroups according to the methods conventionally utilized for amideformations and ester reductions. For example, the carboxylic ester issaponified with aqueous lithium hydroxide. The acid is activated withethyl chloroformate and reduced with sodium borohydride to generate,after removal of the protecting group, the hydroxymethyl phosphonateanalog. The acid is also converted to its acyl chloride and then reactedwith ethylamine to afford the amide analog.

The aryl acetaldehyde is coupled with 2-(diethoxyphosphoryl)-but-3-enoicacid ethyl ester to generate the 2-vinyl substituted ester, according toa procedure such as that reported in Synthesis, 1999, 282. The 2-vinylgroup is converted to the 2-cyclopropyl group under cyclopropanationconditions such as those described in Tetrahedron Lett. 1998, 39, 8621.The ester is converted to the alcohol, which, optionally, can be furthersubjected to reactions such as that described below to generate variousphosphonate-containing mycophenolic acid analogues.

The allylic alcohol is treated with bromomethylphosphonic aciddiisopropyl ester in the presence of a base such as lithium t-butoxidein a solvent such as dimethylformamide. The phenol protecting group isthen removed by treatment with trifluoroacetic acid.

The phenacetaldehyde can alternatively be converted to the allylphosphonium salt, according to a procedure such as that reported in J.Org. Chem. 1987, 52, 849. The phosphonium salt is then treated with thecommercially available 3,3,3-trifluoro-2-oxo-propionic acid ethyl esterand a base such as sodium hydride to generate the 2-trifluoromethylsubstituted ester. The ester is converted to the alcohol, which,optionally, can be further subjected to reactions described earlier togenerate mycophenolic acid analogues with various side chains containingthe phosphonate group.

Example 204 Preparation of Representative Compounds of the InventionIntroduction of R₄ Variants of Examples 200-203:

The enone (synthesis reviewed in Tetrahedron, 1985, 41, 4881-4889) andthe diene (Chem. Pharm. Bull., 1989, 37, 2948-2951) are dissolved in anorganic solvent such as toluene, stirred at room temperature for 24hours and heated to reflux for additional 5 hours, according to aprocedure from J. Med. Chem., 1996, 39, 4181-4196. The reaction mixtureis cooled to room temperature and the solvent removed in vacuo. Thecrude reaction product is further purified by chromatography.

The product of step one is dissolved in an organic solvent such as DCMand m-chloroperbenzoic acid is added, according to a procedure from J.Med. Chem., 1996, 39, 4181-4196. At the end of the reaction, thesolution is poured into aqueous sodium hydrogen sulfite solution. Theorganic layer is washed with saturated aqueous sodium bicarbonatesolution and is dried over sodium sulfate. Filtration and evaporation ofsolvents yields the crude product.

The crude product is dissolved in an organic solvent such as toluene andtreated with dichlorodicyanoquinone (DDQ), according to a procedure fromJ. Med. Chem., 1996, 39, 4181-4196. At the end of the reaction thesolvent is removed in vacuo and the crude material is further purifiedby chromatography.

The product is dissolved in an organic solvent such as DCM and treatedwith boron trichloride at reflux temperature, according to a modifiedprocedure from J. Med. Chem., 1996, 39, 46-55. At the end of thereaction the solution is washed with aqueous HCl solution. The solutionis dried over sodium sulfate. Removal of the solvent yields the crudereaction product. Further purification is achieved by chromatography.

The product of the previous step and triphenylphosphine are dissolved inan organic solvent such as tetrahydrofuran (THF).Diisopropylazodicarboxylate (DIAD) is added dropwise at 0° C. Stirringis continued. A solution of 2-trimethylsilyl ethanol in THF is added andstirring is continued. At the end of the reaction the solvent is removedin vacuo. The crude reaction solid is extracted with a mixture oforganic solvents such as hexanes and diethylether. The washings arecombined and the solvents removed in vacuo. The desired TMS protectedproduct is further purified and separated from the undesired regioisomerby chromatography.

The starting material is dissolved in an organic solvent such asdimethylformamide (DMF) and reacted with N-chlorosuccinimide, accordingto a procedure from J. Med. Chem., 1996, 39, 4181-4196. After thestarting material is consumed the reaction mixture is poured into waterand the product is extracted with diethyl ether. The combined organiclayers are dried over sodium sulfate. Filtration and evaporation of thesolvents yields the crude chloro-substituted product. Furtherpurification is achieved by chromatography.

The starting material is dissolved in an organic solvent such as benzeneand reacted with dimethyl sulfoxide (DMSO), dicyclohexylcarbodiimide(DCC), and orthophosphoric acid according to a procedure from J. Am.Chem. Soc., 1966, 88, 5855-5866. At the end of the reaction, thesuspension is filtered and the organic layer washed with aqueous sodiumbicarbonate solution and dried over sodium sulfate. Filtration andevaporation of solvents yields the crude material. Further purificationis achieved by chromatography.

The product of step one is dissolved in an organic solvent such as DCMor THF and treated with Raney nickel, according to procedures reviewedin Chem. Rev., 1962, 62, 347-404. When all starting material isconsumed, the reaction is filtered and the solvent removed in vacuo.Further purification of the dimethyl substituted product is achieved bychromatography.

The starting material is dissolved in an organic solvent such as DCM andbromine is added, according to a procedure from J. Med. Chem., 1996, 39,4181-4196. At the end of the reaction additional DCM is added and thesolution washed with aqueous sodium thiosulfate solution and brine. Theorganic layer is dried over sodium sulfate. Filtration and evaporationof solvents yields the crude material. Further purification is achievedby chromatography on silica gel.

The product of step one, lithium chloride, triphenylarsine,tributylvinyltin, andtris(dibenzylideneacetone)dipalladium(0)-chloroform adduct are heated inan organic solvent such as N-methylpyrrolidinone at an elevatedtemperature of approximately 55° C., according to a procedure from J.Med. Chem., 1996, 39, 4181-4196. At the end of the reaction the mixtureis cooled to room temperature and poured into a mixture of ice,potassium fluoride, water, and ethyl acetate. Stirring is continued for1 hour. The suspension is filtered through Celite and extracted withethyl acetate. The combined organic extracts are dried over sodiumsulfate. The solvents are removed in vacuo and the crude material isfurther purified by chromatography.

The product of step two is dissolved in a mixture of organic solventssuch as benzene and ethyl acetate. Tris(triphenylphosphine)rhodium(I)chloride is added and the reaction is placed under an atmosphere ofhydrogen, according to a procedure from J. Med. Chem., 1996, 39,4181-4196. The solvents are removed in vacuo and the crude reaction isfiltered through silica gel. Further purification of the 6-ethylsubstituted compound is achieved by chromatography.

The starting material is dissolved in an organic solvent such as DMF.Potassium carbonate and allyl bromide are added and stirring at roomtemperature is continued, according to a procedure from J. Med. Chem.,1996, 39, 4181-4196. After all the starting material is consumed,aqueous HCl solution and diethyl ether are added and the organic layeris collected and the solvent is removed in vacuo.

The crude material from step one is dissolved in N,N-diethylaniline andthe reaction mixture is heated at an elevated temperature of ca. 180° C.At the end of the reaction the mixture is cooled to room temperature andpoured into a mixture of aqueous HCl (2N) and ethyl acetate. The organiclayer is washed with aqueous HCl (2N) and dried over sodium sulfate.Filtration and removal of the allyl compound and solvents yields thecrude product. Further purification is achieved by chromatography.

The product of step 2 is dissolved in a mixture of organic solvents suchas methanol, DCM, and pyridine. The solution is cooled to −78° C. andozone is bubbled into the solution until a blue color persists. Theexcess ozone is removed with a nitrogen stream. The reaction mixture iswarmed to room temperature and thiourea is added. Stirring at roomtemperature is continued. The reaction mixture is filtered andpartitioned between DCM and water. The aqueous layer is extracted withDCM and the combined organic layers are washed with HCl (1 N), saturatedaqueous sodium bicarbonate solution and brine. The solution is driedover sodium sulfate. Filtration and evaporation of the solvents yieldsthe crude aldehyde. Further purification is achieved by chromatography.

The aldehyde is dissolved in an organic solvent such as THF and isreacted with triphenylphosphonium sec-propyl bromide and potassiumtert-butoxide, according to procedures reviewed in Chem. Rev., 1989, 89,863-927. At the end of the reaction the solvent is removed in vacuo andthe crude material purified by chromatography to give the2-methylbut-2-enyl derivative.

Example 205 Preparation of Representative Compounds of FormulaIntroduction of Linkers to Phosphonates:

Representative compounds of the invention can be prepared as illustratedabove. The phenols shown in herein may optionally be alkylated with thereagent of choice. Optionally, the phosphonate moiety will be part ofsuch a reagent; alternatively it will be introduced in a subsequent stepby a variety of means, of which three are illustrated above. Forexample, an alkyl halide may be heated with triethylphosphite in asolvent such as toluene (or other Arbuzov reaction conditions: seeEngel, R., Synthesis of Carbon-Phosphorus Bonds, CRC Press, 1988).Alternatively, an epoxide may be reacted with the anion of a dialkylphosphinate. In a further example, the phosphonate reagent may be theelectrophile; for example, an acetylide anion may be condensed withphosphorus oxychloride and the intermediate dichlorophosphonate quenchedwith ethanol to generate the diethyl ester of the desired phosphonicacid.

Example 206 Preparation of Representative Compounds of Formula 81

[4-(6-Ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phosphonicacid: this product was prepared using methods similar to those describedfor Examples 156 and 181. MS (negative mode): 369.3 [M⁺−1].

Example 207 Preparation of Representative Compounds of Formula 81

2-{[4-(6-Ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enyloxymethyl]-phenoxy-phosphinoylamino}-propionicacid ethyl ester: using methods similar to those described for Example165, the desired product was prepared starting from material analogousto the compound of Example 193. MS (positive mode): 546.3 [M⁺+1] & 568.3[M⁺+Na]

Example 208 Preparation of Representative Compounds of Formula 81

2-({2-[4-(6-Ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-ethyl}-phenoxy-phosphinoylamino)-propionicacid ethyl ester: this product was prepared using methods analogous tothose described for Examples 173 and 193, using2-[(2-amino-ethyl)-phenoxy-phosphinoylamino]-propionic acid ethyl esterin the reductive amination step. MS (positive mode): 559.4 [M⁺+1] &581.3 [M⁺+Na].

Example 209 Preparation of Representative Compounds of Formula 81

2-((1-Ethoxycarbonyl-ethylamino)-{2-[4-(6-ethyl-4-hydroxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2-methyl-but-2-enylamino]-ethyl}-phosphinoylamino)-propionicacid ethyl ester: this product was prepared by methods analogous tothose described for Examples 183, 184, and 187 using2-[(2-aminoethyl)-(1-ethoxycarbonyl-ethylamino)-phosphinoylamino]-propionicacid ethyl ester in the reductive amination step. MS (positive mode):582.4 [M⁺+1] & 604.3 [M⁺+Na].

Example 210 Preparation of Representative Compounds of Formulae 87 and88

Compound 210.1

Synthesis of 210.1: To a solution of 3′-deoxyuridine (995 mg, 4.36 mmol)in 8 mL of anhydrous pyridine was added t-butyldiphenylsilyl chloride(TBDPS-Cl, 1.38 g, 5.01 mmol), and 4-dimethylaminopyridine (DMAP, 27 mg,0.22 mmol). The mixture was stirred at 23° C. for 14 hours and thencooled to 0° C. in a ice-water bath. To this mixture was added benzoylchloride (735 mg, 0.61 mL, 5.2 mmol). The mixture was warmed to 23° C.and stirred for another 2 hours. The mixture was concentrated in vacuoto give a paste, which was partitioned between water and ethyl acetate.The aqueous later was extracted once with ethyl acetate. The combinedethyl acetate layer was washed sequentially with 1 M aqueous citricacid, saturated sodium bicarbonate, and brine. It was dried overanhydrous sodium sulfate and concentrated in vacuo to give a crudeproduct as a yellow oil. Purification by silica gel chromatography(15-65% ethyl acetate in hexane) gave a colorless oil. Yield 1.35 g(54%). ¹H NMR (DMSO-d6): δ 11.38 (s, 1H), 8.01 (d, J=7.9 Hz, 2H), 7.77(d, J=8.2 Hz, 1H), 7.70-7.40 (m, 13H), 5.99 (s, 1H), 5.58 (m, 1H), 7.34(d, J=8.2 Hz, 1H), 4.47 (m, 1H), 4.03 (m, 1H), 3.84 (m, 1H), 2.43 (m,1H), 2.21 (m, 1H), 1.03 (s, 9H) ppm. MS (m/z) 571.1 (M+H⁺), 593.3(M+Na⁺).

Compound 210.2

Synthesis of 210.2: To a solution of 210.1 (1.31 g, 2.3 mmol) in 5 mL ofanhydrous N,N-dimethylformamide was added benzyl chloromethyl ether(0.54 g, 3.45 mmol), N,N-diisopropylethylamine (446 mg, 0.60 mL, 3.45mmol). The mixture was stirred at 23° C. for 4 hours. Water was added.The mixture was extracted with ethyl acetate. The organic layer waswashed sequentially with 1 M aqueous citric acid, saturated sodiumbicarbonate, and brine. It was dried over anhydrous sodium sulfate andconcentrated in vacuo to give a crude product as a yellow oil, which wasused in the next step without further purification.

The crude product obtained above was dissolved in 9 mL of THF. Thesolution was cooled to 0° C. A 1 M solution of TBAF (4.6 mL, 4.6 mmol)was added via syringe. The mixture was warmed to 23° C. and stirred foranother 2 hours. An additional 2.3 mL of 1 M TBAF was added. The mixturewas stirred for another 2 hours at 23° C. Saturated aqueous ammoniumchloride was added to the solution. The mixture was evaporated in vacuoto remove most of THF. The aqueous phase was extracted with ethylacetate. The aqueous layer was washed with brine. It was then dried overanhydrous sodium sulfate and concentrated in vacuo to give a crudeproduct as a yellow oil. Purification by silica gel chromatography(30-80% ethyl acetate in hexane) gave a white solid. Yield of 210.2: 805mg (77% for two steps). ¹H NMR (DMSO-d6): δ 8.04 (m, 3H), 7.67 (t, J=7.3Hz, 1H), 7.55 (t, J=7.6 Hz, 2H), 7.30 (m, 5H), 5.98 (s, 1H), 5.78 (d,J=7.9 Hz, 1H), 5.55 (m, 1H), 5.31 (s, 2H), 5.22 (m, 1H), 4.57 (s, 2H),4.41 (m, 1H), 3.80 (m, 1H), 3.60 (m, 1H), 2.31 (m, 1H), 2.15 (m, 1H)ppm. MS (m/z) 453.1 (M+H⁺), 475.3 (M+Na⁺).

Compound 210.3

Synthesis of 210.3: To a solution of 210.2 (800 mg, 1.77 mmol) in 3.5 mLof a 1:1 mixture of acetonitrile/water was added iodobenzene diacetate(1.25 g, 3.89 mmol), and TEMPO (55 mg, 0.35 mmol). The mixture wasstirred at 23° C. for 14 hours. The mixture was then froze in a −78° C.bath and lyophilized to give a solid residue. This residue was purifiedby silica gel chromatography (0-15% methanol in dichloromethane).Product 210.3 was obtained as a white solid. Yield: 735 mg (89%). ¹H NMR(DMSO-d6): δ 8.13 (d, J=7.6 Hz, 1H), 8.03 (d, J=7.7 Hz, 2H), 7.68 (m,1H), 7.58 (t, J=7.0 Hz, 2H), 7.29 (m, 5H), 6.04 (s, 1H), 5.85 (d, J=8.3Hz, 1H), 5.62 (m, 1H), 5.31 (s, 2H), 4.87 (m, 1H), 4.58 (s, 2H),2.40-2.20 (m, 2H) ppm. MS (m/z) 467.1 (M+H⁺), 489.3 (M+Na⁺).

Compound 210.4

Synthesis of 210.4: To a deoxygenated solution of 210.3 (730 mg, 1.57mmol) and pyridine (0.51 mL, 6.26 mmol) in 7 mL of anhydrous DMF, wasadded lead tetraacetate (3.47 g, 7.83 mmol). The mixture was stirred at23° C. for 14 hours shielded from light. The mixture was diluted with 15mL of ethyl acetate and 10 mL of water. This mixture filtered through apad of Celite and separated. The aqueous phase was extracted withanother 10 mL of ethyl acetate. The combined ethyl acetate extract waswashed with brine, dried over sodium sulfate, and evaporated in vacuo togive the crude product as an oil. The crude product 210.4 was purifiedby silica gel chromatography (10-50% ethyl acetate in hexane). Productsof two diastereomers were obtained as a white foam. Yield: 400 mg (53%).¹H NMR (DMSO-d6): δ 8.01 (m, 2H), 7.82-7.63 (m, 2H), 7.57 (m, 2H), 7.31(m, 5H), 6.58 (m, 1H), 6.17 (m, 1H), 5.83 (m, 1H), 5.65 (m, 1H), 5.31(s, 2H), 4.59 (s, 2H), 2.76 and 2.28 (m, 1H), 2.10 (m, 1H), 2.07 (s, 3H)ppm. MS (m/z) 481.0 (M+H⁺), 503.3 (M+Na⁺).

Compound 210.5a and 210.5b

Synthesis of 210.5a: To a solution of 210.4 (300 mg, 0.63 mmol) in 6 mLof anhydrous dichloromethane was added diethyl hydroxymethyl-phosphonate(0.37 mL, 2.5 mmol), followed by trimethylsilyltrifluoromethanesulfonate (0.34 mL, 1.88 mmol). The mixture was stirredat 23° C. for 6 hours. Triethylamine (0.44 mL, 3.15 mmol) was added,followed by water. The mixture was extracted with ethyl acetate. Theorganic layer was washed with 1 M aqueous citric acid, saturated sodiumbicarbonate, and brine. It was then dried over anhydrous sodium sulfate,and evaporated in vacuo to give a residue. This crude product waspurified by silica gel chromatography (75-95% ethyl acetate in hexane)to give two products, which were diastereomers of each other shown above(210.5a and 210.5b). Yield of 210.5a: 53 mg (14%). Yield of 210.5b: 129mg (35%).

Analytical data for 210.5a: ¹H NMR (Acetonitrile-d3): δ 8.04 (d, J=7.0Hz, 2H), 7.77 (d, J=7.9 Hz, 1H), 7.69 (t, J=7.5 Hz, 1H), 7.53 (m, 2H),7.33 (m, 5H), 6.38 (d, J=4.0 Hz, 1H), 5.80 (d, J=8.2 Hz, 1H), 5.63 (m,1H), 5.52 (m, 1H), 5.41 (s, 2H), 4.64 (s, 2H), 4.17 (m, 4H), 4.08 (dd,J=13.8, 10.1 Hz, 1H), 3.92 (dd, J=13.7, 9.5 Hz, 1H), 2.66-2.42 (m, 2H),1.35 (t, J=7.0 Hz, 6H) ppm. MS (m/z) 589.2 (M+H⁺), 611.3 (M+Na⁺).Stereochemistry of 210.5a was confirmed by additional 2D NMRexperiments.

Analytical data for 210.5b: ¹H NMR (Acetonitrile-d3): δ 8.08 (d, J=7.3Hz, 2H), 7.69 (t, J=7.5 Hz, 1H), 7.55 (m, 2H), 7.43 (d, J=8.2 Hz, 1H),7.36 (m, 5H), 6.11 (d, J=2.4 Hz, 1H), 5.77 (d, J=8.3 Hz, 1H), 5.57 (m,2H), 5.41 (s, 2H), 4.66 (s, 2H), 4.12 (m, 5H), 3.88 (dd, J=14.0, 5.2 Hz,1H), 2.82 (m, 1H), 2.25 (m, 1H), 1.27 (t, J=7.0 Hz, 6H) ppm. MS (m/z)589.0 (M+H⁺), 611.2 (M+Na⁺).

Compound 210.6

Synthesis of 210.6: To a solution of 210.5a (110 mg, 0.19 mmol) in 3 mLof acetonitrile was added 2,6-lutidine (0.43 mL, 3.74 mmol), followed byiodotrimethylsilane (0.53 mL, 3.74 mmol). After stirring at 23° C. for30 min, the mixture was heated to 40° C. and stirred at that temperaturefor another 4 hours. The reaction mixture was cooled to 23° C.Triethylamine (0.52 mL, 3.74 mmol) was added, followed by water (10 mL).The aqueous mixture was extracted twice with 5 mL of diethyl ether. Theresulting aqueous solution was frozen in a −78° C. bath and waslyophilized to give a yellow solid. This crude product was purified byreversed phase HPLC to give 210.6 as a light yellow solid. Yield 26 mg(34%). MS (m/z) 411.3 (M−H⁻).

Compound 210.7

Synthesis of 210.7: Phosphonate 210.6 (12 mg, 0.029 mmol),carbonyldiimidazole (47 mg, 0.29 mmol), and tri-n-butylamine (5.4 mg,0.029 mmol) were dissolved in 0.3 mL of anhydrous dimethylformamide(DMF). The mixture was stirred at 23° C. for 4 hours. MeOH (0.020 mL)was added and the mixture was stirred for another 30 minutes. A solutionof tributylammonium pyrophosphate (159 mg, 0.29 mmol) in 0.63 mL ofanhydrous DMF was added. The resulting mixture was stirred at 23° C. for14 hours. The mixture was evaporated in vacuo to remove most of the DMF.The residue was dissolved in 5 mL of water and was purified byion-exchange chromatography (DEAE-cellulose resin, 0-50%triethylammonium bicarbonate in water) to give a white solid, which wasused directly in the next reaction.

The product obtained above was dissolved in 2 mL of water. A 0.3 mL of a1 M solution of sodium hydroxide in water was added. The mixture wasstirred at 23° C. for 40 min. Acetic acid was added to adjust the pH ofthe solution to 5. The solution was diluted with water and purified withan ion-exchange column (DEAE-cellulose resin, 0-50% triethylammoniumbicarbonate in water) to give diphosphophosphonate 210.7 as a whitesolid, which is the triethylammonium salt of the structure shown above.Yield 10 mg (45% for two steps). ¹H NMR (D₂O): δ 7.79 (d, J=7.6 Hz, 1H),5.89 (m, 1H), 5.85 (d, J=7.6 Hz, 1H), 5.41 (m, 1H), 4.49 (m, 1H),4.02-3.65 (m, 2H), 3.06 (m, 18H), 2.20 (m, 2H), 1.14 (m, 27H) ppm. ³¹PNMR (D₂O): δ 7.46 (d, 1P), −9.45 (d, 1P), −23.11 (t, 1P) ppm. MS (m/z)467.0 (M−H⁻).

Compound 210.8

Synthesis of 210.8: To a solution of 210.6 (16 mg, 0.039 mmol) in 0.4 mLof water was added NaOH (7.8 mg, 0.19 mmol). The solution was stirred at23° C. for 1 hour. Acetic acid (0.012 mL) was added to the solution. Themixture was then purified by reversed phase HPLC (eluted with 100%water) to give 4.6 mg of 210.8 as a white solid (38% yield). ¹H NMR(D₂O): δ 7.83 (d, J=8.3 Hz, 1H), 5.86 (d, J=3.4 Hz, 1H), 5.82 (d, J=7.9Hz, 1H), 4.48 (m, 1H), 3.68 (m, 1H), 3.37 (m, 1H), 2.16 (m, 2H) ppm. ³¹PNMR (D₂O): δ 12.60 (s, 1P) ppm. MS (m/z) 615.1 (2M−H⁻).

Example 211 Preparation of Representative Compounds of Formula 89

Representative compounds of the invention can be prepared as illustratedabove. Analogs attached to the 4-position of levamisole of the type211.2 are prepared from the R³-protected starting material 211.3.Following resolution of the enantiomers, the protecting group is cleavedand the appropriate phosphonate is alkylated on to the phenol 211.5.

As an example, the racemic methyl ether 211.6 is synthesized by apublished procedure (J. Med. Chem. 1966, 9, 545-551). The desiredenantiomer 211.7 is resolved between a chiral solid and an achiralsupercritical fluid phase (Tetrahedron: Asymmetry 1999, 10, 1275-1281).After reaction with BBr₃, phenol 211.5 is furnished. Final alkylationwith diethyl phosphomethyltriflate affords the desired phosphonate211.8.

Example 212 Preparation of Representative Compounds of Formulae 90 and93

Representative compounds of the invention can be prepared as illustratedabove. Triol 212.2 can be selectively alkylated with an appropriatephosphonate-containing alkylating agent. Deprotection of nitrogenaffords phosphonate 212.1.

A specific compound of Formula 90 or 93 can be prepared as illustratedabove. The Boc-protected(1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol, compound212.3, is prepared by stirring the(1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (WO 99/19338and Evans, G. B. et al., Tetrahedron, 2000, 56, 3053, also reported inEvans, G. B. et al., J. Med. Chem. 2003, 46, 3412) with BOC anhydride asdescribed in Greene, T., Protective Groups In Organic Synthesis,Wiley-Interscience, 1999. Compound 212.3 is then treated in a solventsuch as tetrahydrofuran or dimethylformamide with a base such as sodiumhydride. When bubbling ceases, diethyl phosphonomethyltriflate (preparedaccording to Tetrahedron Lett., 1986, 27, 1477) is added, yielding thedesired phosphonate 212.4 after deprotection of the BOC group usingtrifluoroacetic acid (TFA).

Example 213 Preparation of Representative Compounds of Formulae 91, 92,94, and 95

Representative compounds of the invention can be prepared as illustratedabove. The primary alcohol of triol 213.1 can be selectively alkylatedwith an appropriate phosphonate-containing alkylating agent.Deprotection of nitrogen affords phosphonates 213.2 and 213.3.

Specific compounds of Formulae 91, 92, 94, and 95 can be prepared asillustrated above. The Boc-protected(1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol, compound213.4, is prepared by stirring the(1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (WO 99/19338and Evans, G. B. et al., Tetrahedron, 2000, 56, 3053, also reported inEvans, G. B. et al., J. Med. Chem. 2003, 46, 3412) with BOC anhydride asdescribed in Greene, T., Protective groups in organic synthesis,Wiley-Interscience, 1999. Subsequent protection of the primary alcoholusing a TBS group can be achieved using TBSCl and imidazole in solventssuch as CH₂Cl₂ as described in Greene, Protective Groups In OrganicSynthesis, Wiley-Interscience, 1999, to provide compound 213.4. Compound213.4 is then treated in a solvent such as tetrahydrofuran ordimethylformamide with a base such as sodium hydride. When bubblingceases, diethyl phosphonomethyltriflate (prepared according toTetrahedron Lett., 1986, 27, 1477) is added, yielding a mixture of thedesired phosphonate diester 213.5 and 213.6 after deprotection of theBOC group using trifluoroacetic acid (TFA). Compounds 213.5 and 213.6can be also prepared via a more complicated 2′-OH protected analog of213.4 followed by alkylation using the diethyl phosphonomethyltriflateto provide compound 213.5 exclusively. Compound 213.6 can also beprepared by installation of a different protecting group at the 3′-OHposition, followed by deprotection of 2′-OH and alkylation with diethylphosphonomethyltriflate at the 2′-center followed by globaldeprotection.

Example 214 Preparation of Representative Compounds of Formula 96

Representative compounds of the invention can be prepared as illustratedabove. The desired phosphonate substituted analogs are prepared byreaction of intermediate 214.5 (obtained as described in U.S. Pat. No.5,464,826) with the respective alkylating reagents 214.6. Illustratedabove is the preparation of phosphonate linkage to2′2′-difluoronucleosides through the 5′-hydroxyl group. Theappropriately protected base as described in U.S. Pat. No. 5,464,826 isdissolved in a solvent such as DMF, THF and is treated with aphosphonate reagent bearing a leaving group, for example, bromine,mesyl, tosyl, or trifluoromethanesulfonyl in the presence of a suitableorganic or inorganic base.

For instance, 214.1 (obtained as described in U.S. Pat. No. 5,464,826)dissolved in DMF, is treated with two equivalents of sodium hydride andone equivalent of (toluene-4-sulfonylmethyl)-phosphonic acid diethylester 214.8, prepared according to the procedures in J. Org. Chem. 1996,61, 7697, to give the corresponding phosphonate 214.9 in which thelinkage is a methylene group. Using the above procedure but employingdifferent phosphonate reagents 214.6 in place of 214.8 the correspondingproducts 214.2 bearing different linking groups are obtained.

Example 215 Preparation of Representative Compounds of Formula 97

Representative compounds of the invention can be prepared as illustratedabove. Compounds 215.5 containing a variety of suitably protected basesas cited and prepared according to U.S. Pat. No. 5,464,826, can beconverted to glycal 215.10 according to the process reported in J. Am.Chem. Soc. 1972, 94, 3213. Glycal 215.10 is then treated with IBr in thepresence of alcohol 215.11 to provide intermediate 215.12 (see J. Org.Chem. 1991, 56, 2642). The iodide of intermediate 215.12 can be treatedwith AgOAc to provide the corresponding acetate, which is deacetylatedin the presence of catalytic sodium methoxide in methanol. Treatment ofthe resulting alcohol with DEAD and PPh₃ in the presence of acetic acid,followed by another deprotection with catalytic sodium methoxide inmethanol will provide intermediate 215.3. The phosphonates ofintermediates 215.3 can then be converted into their final desiredforms.

For instance, glycal 215.14 is prepared according to the procedurescited above (U.S. Pat. No. 5,464,826; J. Am. Chem. Soc. 1972, 94, 3213).Glycal 215.14 is then treated with IBr in the presence of diethylphosphonomethanol, 215.8, to provide intermediate 215.15 (see J. Org.Chem. 1991, 56, 2642). Intermediate 215.15 is then treated with AgOAcfollowed by deprotection with catalytic NaOMe in MeOH. This compound isthen converted into 215.16 by a Mitsunobu reaction with DEAD/PPh₃ andHOAc in THF, followed by a second catalytic NaOMe/MeOH deprotection.Base conversion of uracil to cytosine is carried out prior to the acetyldeprotection using the procedures in Bioorg. Med. Lett. 1997, 7, 2567.At any point in the synthesis sequence where it is appropriate, thephosphonate group may be converted into the phsophonate with the desiredsubstitution. Using the above procedure but employing differentphosphonate reagents 215.11 in place of 215.8 the corresponding products215.3 bearing different linking groups are obtained.

Example 216 Preparation of Representative Compounds of Formula 98

Representative compounds of the invention can be prepared as illustratedabove. The phosphorus containing merimepodib analog 216.2 is synthesizedfrom parent compounds by alkylation. Merimepodib (216.1) is obtained bythe procedure as described in U.S. Pat. No. 6,054,472 and U.S. Pat. No.6,344,465. Shown above is the procedure for the synthesis of 216.2.Methoxy group of merimepodib (216.1) is demethylated to the phenolic OHusing a suitable reagent, such as boron tribromide. The phosphonatemoiety is introduced to the phenolic OH in a suitable aprotic solventsuch as, DMF by treatment with the phosphonate reagent 216.7, bearing aleaving group, for example, bromine, mesyl, tosyl, ortrifluoromethanesulfonyl, in the presence of a suitable organic orinorganic base.

For instance, a solution of 216.1 in dichloromethane is treated withboron tribromide to obtain the demethylated compound 216.8. Compound216.8 is then treated with cesium carbonate and one equivalent of(trifluoromethanesulfonyloxy)methylphosphonic acid diethyl ester 216.9to give merimepodib-phosphonate 216.10 in which the linkage is amethylene group as shown above. Using the above procedure but employingdifferent phosphonate reagents 216.7, the corresponding products 216.2bearing different linking group can be obtained.

Example 217 Preparation of Representative Compounds of Formulae 99 and100

Representative compounds of the invention can be prepared as illustratedabove. The imidazole containing intermediate 217.13 is synthesized froman aldehyde 217.12 by the procedure of Shih in Tetrahedron Lett. 1993,34, 595. Compound 217.12 is prepared by a two-step procedure describedin U.S. Pat. No. 5,807,876, U.S. Pat. No. 6,054,472, and U.S. Pat. No.6,344,465. The imidazole is protected using suitable reagent, forexample 2-(trimethylsilyl)ethyoxymethyl (SEM) chloride, and the compound217.14 is converted to 217.15 by the similar procedure described for thesynthesis of 216.1 in U.S. Pat. No. 6,054,472 and U.S. Pat. No.6,344,465. After the protecting group on the imidazole of 217.15 isremoved, the phosphonate containing moiety is introduced to theimidazole as shown below.

For example, compound 217.15 is treated with tetrabutylammonium fluoridein THF under refluxing conditions and the resulting 217.16 is alkylatedwith 217.9 using sodium hydride as a base to obtain the two isomers217.17 and 217.18, which are separated by chromatography.

Example 218 Preparation of Representative Compounds of Formula 101

Representative compounds of the invention can be prepared as illustratedabove. Illustrated above is the preparation of merimepodib analog 218.5.Tetrasubstituted benzene derivatives are obtained by literatureprocedures (Ichikawa and Ichibagase Yakugaku Zasshi 1963, 83, 103;Norio, A. et al. Tetrahedron Lett. 1992, 33(37), 5403). After thephenolic OH is protected with a suitable protecting group, for examplebenzyl group, the compound 218.21 is synthesized by the same procedureof the synthesis of 216.1 as described in U.S. Pat. No. 6,054,472, andU.S. Pat. No. 6,344,465. After the protecting group is removed, thephosphonate containing moiety is introduced to the phenolic OH using thephosphonate reagent 218.7, bearing a suitable leaving group.

For example, a solution of 218.22, which is obtained by the procedure ofNorio et al. (Tetrahedron Lett. 1992, 33(37), 5403), is treated withsodium hydride and one equivalent of benzyl bromide in DMF to get218.23. Compound 218.23 is converted to 218.24 by a series of stepsreported in U.S. Pat. No. 6,054,472, and U.S. Pat. No. 6,344,465 for thesynthesis of 216.1 from 217.12. After the benzyl protecting group of218.24 is removed by catalytic hydrogenation, a phosphonate bearingmoiety is attached by alkylation of the resulting phenol in DMF usingsodium hydride and one equivalent of(trifluoromethanesulfonyloxy)methylphosphonic acid diethyl ester 218.7to give 218.25.

Example 219 Preparation of Representative Compounds of Formula 102

Representative compounds of the invention can be prepared as illustratedabove. Synthesis of merimepodib analog 219.6 is shown above. Compound219.26, an intermediate in the synthesis of 216.1, is treated withcarbonyldiimidazole or triphosgene followed by the compound 219.27,which has an handle to attach phosphonate moiety. Compound 219.27bearing an extra substituent is synthesized from the tri substitutedphenol with cyano and nitro groups, which is either commerciallyavailable or can be prepared by literature procedures (Zolfigol, M. A.et. al. Indian J. Chem. Sect. B 2001, 40, 1191; De Jongh, R. O. et al.Rec. Trav. Chim. Pays-Bas 1968, 87, 1327). The resulting 219.28 isconverted to 219.29 using the procedure described in U.S. Pat. No.6,054,472, and U.S. Pat. No. 6,344,465 for the synthesis of 216.1. Thephosphonate moiety of 219.6 is attached after deprotection of the benzylgroup of 219.29:

For example, the bromine substituent of compound 219.30 is substitutedwith cyano group by the procedure of De Jongh, R. O. et al. (Rec. Trav.Chim. Pays-Bas 1968, 87, 1327) and the methoxy group is converted tobenzyloxy group as a protecting group, which affords compound 219.31.After selective reduction of cyano to aminomethyl group by borane, theamino group is protected with Boc group and then the reduction of thenitro group using tin (II) chloride generates compound 219.32. Thissubstituted aniline 219.32 is then treated with a reaction mixture ofthe compound 219.26 and carbonyldiimidazole, as described for thesynthesis of 216.1 in U.S. Pat. No. 6,054,472, and U.S. Pat. No.6,344,465, to form the urea 219.33. Compound 219.33 is easily convertedto 219.34, analog of 216.1 bearing benzyloxy group. Deprotection of thebenzyl group using catalytic hydrogenation followed by attachment of aphosphonate moiety using 219.9 in the presence of cesium carbonateproduces 219.34.

Example 220 Synthesis of Exemplary Compounds of the Invention

Appropriate oxidant(s) can convert the primary alcohol (5′-hydroxy)shown in 518-1.3 to a carboxylic acid or its corresponding ester. In thecase of an ester, an additional deprotection step will give thecarboxylic acid, 518-1.4. A variety of oxidation procedures exist in theliterature and can be utilized here. These include but are not limitedto the following methods: (i) pyridinium dichromate in Ac₂O, t-BuOH, anddichloromethane producing the t-butyl ester, followed by a deprotectionusing reagent such as trifluoroacetic acid to convert the ester to thecorresponding carboxylic acid (see Classon, et al, Acta Chem. Scand.Ser. B; 39; 1985; 501-504. Cristalli, et al; J. Med. Chem.; 31; 1988;1179-1183); (ii) iodobenzene diacetate and2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) inacetonitrile, producing the carboxylic acid (See Epp, et al; J. Org.Chem. 64; 1999; 293-295. Jung et al; J. Org. Chem.; 66; 2001;2624-2635); (iii) sodium periodate, ruthenium(III) chloride inchloroform producing the carboxylic acid (see Kim, et al, J. Med. Chem.37; 1994; 4020-4030. Homma, et al; J. Med. Chem.; 35; 1992; 2881-2890);(iv) chromium trioxide in acetic acid producing the carboxylic acid (seeOlsson et al; J. Med. Chem.; 29; 1986; 1683-1689. Gallo-Rodriguez et al;J. Med. Chem.; 37; 1994; 636-646); (v) potassium permanganate in aqueouspotassium hydroxide producing the carboxylic acid (see Ha, et al; J.Med. Chem.; 29; 1986; 1683-1689. Franchetti, et al; J. Med. Chem.; 41;1998; 1708-1715.) (vi) nucleoside oxidase from S. maltophilia to givethe carboxylic acid (see Mahmoudian, et al; Tetrahedron; 54; 1998;8171-8182.)

The preparation of 518-1.5 from 518-1.4 using lead(IV) tetraacetate(LG=OAc) was described by Teng et al; J. Org. Chem.; 59; 1994; 278-280and Schultz, et al; J. Org. Chem.; 48; 1983; 3408-3412. When lead(IV)tetraacetate is used together with lithium chloride (see Kochi, et al;J. Am. Chem. Soc.; 87; 1965; 2052), the corresponding chloride isobtained (1.5, LG=Cl). Lead(IV) tetraacetate in combination withN-chlorosuccinimide can produce the same product (1.5, LG=Cl) (see Wang,et al; Tet. Asym.; 1; 1990; 527 and Wilson et al; Tet. Asym.; 1; 1990;525). Alternatively, the acetate leaving group (LG) can also beconverted to other leaving group such as bromide by treatment oftrimethylsilyl bromide to give 518-1.5 ((see Spencer, et al; J. Org.Chem.; 64; 1999; 3987-3995).

The coupling of 518-1.5 (LG=OAc) with a variety of nucleophiles weredescribed by Teng et al; Synlett; 1996; 346-348 and U.S. Pat. No.6,087,482; Column 54 line 64 to Column 55 line 20. Specifically, thecoupling between 518-1.5 and diethyl hydroxymethylphosphonate in thepresence of trimethylsilyl trifluoromethanesulfonate (TMS-OTf) wasdescribed. It can be envisioned that other compounds with the generalstructure of HO-linker-POR^(P1)R^(P2) can also be used so long as thefunctional groups in these compounds are compatible with the couplingreaction conditions. There are many examples in the published literaturedescribing the coupling of 518-1.5 (LG=halogen) with a variety ofalcohols. The reactions can be facilitated with a number of reagents,such as silver(I) salts (see Kim et al; J. Org. Chem.; 56; 1991;2642-2647, Toikka et al; J. Chem. Soc. Perkins Trans. 1; 13; 1999;1877-1884), mercury(II) salts (see Veeneman et al; Rec. Trav. Chim.Pays-Bas; 106; 1987; 129-131), boron trifluoride diethyl etherate (seeKunz et al; Hel. Chim Acta; 68; 1985; 283-287), Tin(II) chloride (seeO'Leary et al; J. Org. Chem.; 59; 1994; 6629-6636), alkoxide (seeShortnacy-Fowler et al; Nucleosides Nucleotides; 20; 2001; 1583-1598),and iodine (see Kartha et al; J. Chem. Soc. Perkins Trans. 1; 2001;770-772). These methods can be selectively used in conjunction withdifferent methods in forming 518-1.5 with various leaving groups (LG) toproduce 518-1.6.

The introduction and removal of protecting groups from a compound is acommonly practiced art in organic synthesis. Many sources of informationof the art are available in the published literature, e.g. Greene andWuts, Protecting Groups in Organic Synthesis, 3^(rd) Ed., John Wiley &Sons, Inc., 1999. The main purpose is to temporarily transform afunctional group so that it will survive a set of subsequent reactionprocedures. Afterwards, the original functional group can be restored bya preconceived deprotection procedure. Therefore, the transformationsfrom 518-1.1 to 518-1.2, from 518-1.2 to 518-1.3, and from 518-1.6 to518-A are intended to allow the core components of the transformations(from 518-1.3 to 518-1.6) to occur while preserving the functionalgroups already exist in the compound structures.

The 5′-hydroxyl group of ribavirin (518-2) can be selectively protectedby an appropriate protecting group. The product, 518-3, can be treatedwith benzoyl chloride, an appropriate base, in the presence of catalyticamount of 4-dimethylaminopyridine, to convert 2′- and 3′-hydroxyl groupsto their corresponding benzoyl esters, 518-4. The 5′-hydroxyl group canbe selectively deprotected to give 5. Following procedure described foranalogous compound in U.S. Pat. No. 6,087,482, FIG. 2, 518-4 can beconverted to 518-7 in a three-step sequence. Treating 518-7 with acoupling agent, such as trimethylsilyl trifluoromethanesulfonate, in thepresence of an appropriate alcohol containing a phosphonate group canproduce 518-8. Lastly, treating 518-8 with aqueous sodium hydroxide candeprotect the 2′- and 3′-hydroxyl groups to give 518-1. It is importantto point out that R^(P1) and R^(P2) in 518-8 and 518-1 do not need to bethe same.

A variety of compounds of the general structure 518-1.1 can either beprepared using procedures described in the literature, or be purchasedfrom commercial sources. The following are good sources for informationon the art of preparing a variety of compounds of the general structure518-1.1, Townsend, Chemistry of Nucleosides and Nucleotides, PlenumPress, 1994; and Vorbruggen and Ruh-Pohlenz, Handbook of NucleosideSynthesis, John Wiley & Sons, Inc., 2001. Some exemplary precursors,starting materials and their commercial sources include:

Compound 518-2.1 in Scheme 518-2 is prepared using method described (WO01/90121, page 115, table). The 5′-hydroxyl in 518-2.1 is protected ast-butyldimethylsilyl (TBDMS) ether. The 2′- and 3′-hydroxyl groups canbe protected as benzoyl (Bz) esters to give 518-2.2. The 5′-hydroxyl canthen be deprotected to give 518-2.3. Oxidation using iodobenzenediacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO)convert the primary alcohol to the corresponding acid 518-2.4. Furtheroxidation of 518-2.4 using lead tetraacetate can produce 518-2.5.Coupling between 518-2.5 and diethyl hydroxymethylphosphonate (availablefrom Sigma-Aldrich, Cat. No. 39, 262-6) effected by TMS-OTf can afford518-2.6. Treating 518-2.6 with TMS-Br converts the phosphodiester to thecorresponding phosphonic acid 518-2.7. Deprotection of the 2′- and3′-hydroxyl gives 518-2.8 as an example of the generic structure 518-A,where Base is an adenine, R¹, R⁵, and R⁶ are hydrogen, R² is methylgroup, R³ and R⁴ are hydroxyl groups, linker is a methylene group, andR^(P1) and R^(P2) are both hydroxyl groups.

The phosphonic acids in 518-2.7 and 518-2.8 are used as examples forillustration purpose. Other forms of phosphonates can be access via thephosphonic acid, or other forms, such as the corresponding diesters. Seesection INTERCONVERSIONS OF PHOSPHONATES for details.

Many compounds of the generic structure 518-1.1 with the sugar moiety inits L-configuration are either commercially available or can be preparedby procedures described in the published literature. The oppositeD-configuration enantiomers of the L-nucleoside analogs previouslydiscussed can be prepared from the precursors that are the oppositeenantiomers of 518-3.1, 518-3.2, and 518-3.3. Scheme 518-3 describes thepreparation of the opposite enantiomers of 518-3.1, 518-3.2, and518-3.3.

The commercially available starting material 518-4.1 can be converted to518-4.4, which is the opposite enantiomer of 518-3.1, using the sequenceof reactions outlined above in Scheme 518-3. The osmium tetraoxidecatalyzed dihydroxylation reaction should introduce the diol selectivelyin the opposite face to the tert-butyldimethylsilyl (TBDMS) ether of thehydroxymethyl group. The diol in intermediate 518-4.3 can be protectedas TBDMS ether. Diisobutylaluminum hydride reduction of the lactone atlow temperature should produce 518-4.6, which can be converted to518-4.6 by acetylation. Deprotection of 518-4.6 should produce L-ribose(518-4.7). Acylation reaction can convert all hydroxyl groups in 518-4.7to the corresponding benzoyl esters. Standard coupling reactions with avariety of nucleobases should produce 518-4.10, which is the oppositeenantiomer of 518-3.3.

The synthesis of3-cyano-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-1,2,4-triazole (518-2)is described in US 2002/0156030 A1, page 6, paragraph 0078 to paragraph0079. Using this starting material, one can synthesize carboxamidecompound 518-1 (Scheme 518-4) or formamidine compound 518-1 (Scheme518-5) using the sequences of chemical transformations outlined inSchemes 518-4 and 518-5, respectively.

Appropriate protection, deprotection procedures (See Greene and Wuts,(1999) Protective Groups in Organic Synthesis) can be employed toprepare 518-3, in which the 5′-hydroxyl group is protected, while the2′, and 3′-hydroxyl groups are not (Schemes 518-4 and 518-5). Subsequentprotection, depretection procedures can introduce protecting groups suchas benzoyl group to the 2′- and 3′-hydroxyl, and leave the 5′-hydroxylgroup unprotected as in 518-4. Oxidation can convert the primary alcoholin 518-4 to the corresponding carboxylic acid or its ester. An optionaldeprotection of the ester can give the acid 518-5 as product. Furtheroxidation using oxidant such as lead tetraacetate can convert 518-5 to518-6, in which the leaving group is an acetate. Treating 518-6 with analcohol containing a phosphonate moiety in the presence of appropriatecoupling agent, such as trimethylsilyl trifluoromethanesulfonate, willgive 518-8 as product. Finally, treating 518-8 with the proceduredescribed in US 2002/0156030 A1, page 6, paragraph 0081, should give518-1 as product. It is important to point out that R^(P1) and R^(P2) in518-7, 518-8 and 518-1 do not need to be the same.

A solution of tert-butyl hydroperoxide (t-BuOOH) in benzene (68%, 3 eq)is added dropwise to a solution of allylic alcohol 518-1 (synthesized asdescribed in Tet. Letters (1997) 38:2355-58) and VO(acac)₂ in benzene(final concentration 0.1 M) at room temperature (Scheme 518-6). After 1h of stirring at room temperature, saturated aqueous Na₂S₂O₃ is added tothe reaction mixture. The resulting solution is extracted with EtOAc,washed with H₂O, and dried over sodium sulfate. After removal ofsolvent, the crude product 518-2 is purified by column chromatography onsilica.

Epoxide 518-2 and p-anisylchlorodiphenylmethane (1.5 eq) is dissolved inanhydrous pyridine (0.17 M) and stirred at 25° C. for 2d. Solvents wereremoved under reduced pressure and the residue dissolved in EtOAc. Theorganics were washed with water, saturated aqueous NaHCO₃, and driedover sodium sulfate. After removal of solvent, the crude product 518-3is purified by column chromatography on silica.

To a solution of methyltriphenylphosphonium bromide (2 eq) in anhydrousTHF at −78° C. is added n-butyllithium (2.2 eq). The solution is allowedto warm to room temperature and stirred for 20 min. After recooling to−78° C., this solution is added to fully protected epoxide 518-3 in THF(final concentration 0.06 M). The reaction mixture is allowed to warm toroom temperature and stirred for 12 h at which point H₂O is added andextracted with diethyl ether. The combined organics were dried oversodium sulfate. After removal of solvent, the crude product 518-4 ispurified by column chromatography on silica.

Sodium hydride (1 eq) and 2-amino-4-chloro-7H pyrrolo[2,3-d]pyrimidine(1 eq) were dissolved in anhydrous DMF (0.06 M) and stirred at 120° C.for 10 min. A solution of 518-4 in DMF is then added and the reactionmixture is stirred 12 h at 120° C. at which point the solvents wereevaporated under reduced pressure. The residue is dissolved in CH₂Cl₂,washed with H₂O, and dried over sodium sulfate. After removal ofsolvent, the crude product 518-5 is purified by column chromatography onsilica.

Compound 518-5 is dissolved in dichloromethane and added to a solutionof 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one(Aldrich, Dess-Martin periodinane, 4 eq) in dichloromethane (finalconcentration 0.06 M). The reaction mixture is stirred at roomtemperature for 4 d at which point it is diluted with EtOAc and pouredinto a solution of sodium thiosulfate in saturated aqueous sodiumbicarbonate solution. The organic layer is separated and dried oversodium sulfate. After removal of solvent, the crude product 518-6 ispurified by column chromatography on silica.

A solution of ketone 518-6 in anhydrous THF is added to a solution ofmethylmagnesium bromide (4 eq) in anhydrous THF (0.1 M) at −78° C. Thereaction mixture is stirred for 12 h at −60° C. at which point thereaction is quenched with saturated aqueous NH₄Cl solution. The mixtureis filtered over celite and washed with EtOAc. The combined organicswere washed with saturated aqueous NH₄Cl, water and dried over sodiumsulfate. After removal of solvent, the crude product 7 is purified bycolumn chromatography on silica.

A solution of alcohol 518-7 in anhydrous THF (0.06 M) is treated with asolution of tetrabutylammonium fluoride (1.5 eq) in THF at roomtemperature. The reaction mixture is stirred for 3 h at which point thesolvents were evaporated. The crude desilylated diol 518-8 is purifiedby column chromatography on silica.

To a solution of diol 518-8 and benzenesulfonic aciddiisopropoxy-phosphorylmethyl ester (1.2 eq) in anhydrous DMF (0.1 M) isadded magnesium tert-butoxide (1 eq). The reaction mixture is heated to80° C. for 12 h. After cooling to room temperature, 1 N citric acid isadded and extracted with EtOAc. The organics were neutralized withsaturated aqueous NaHCO₃, washed with saturated aqueous NaCl, and driedover sodium sulfate. After removal of solvent, the crude product 518-9is purified by column chromatography on silica.

Compound 518-9 is dissolved in 80% acetic acid and stirred 12 h at roomtemperature. After removal of solvent, the crude product 518-10 ispurified by column chromatography on silica.

Phosphonate ester 518-10 and 2,6-lutidine (8 eq) is dissolved in CH₃CNand treated with trimethylsilyliodide (8 eq). After stirring for 3 h atroom temperature, triethylamine (8 eq) is added followed by methanol.After removal of solvent, the crude product 518-11 is purified by columnchromatography on silica.

Phosphonic diacid 518-11 is dissolved in 1,4-dioxane and treated with 4N NaOH and heated to 100° C. for 4 h. After cooling to room temperature,the reaction mixture is neutralized with 4N HCl. After removal ofsolvent, the crude product is purified by column chromatography onsilica to provide 518-12.

Compound 518-13 (Paquette et al J. Org. Chem. (1997) 62:1730-1736) istreated with p-methoxybenzyl bromide (1.5 eq.), sodium hydride (1.4 eq)in dry DMF at room temperature (Scheme 518-7). The reaction is monitorby TLC for the disappearance of 518-13. The reaction is quenched by theaddition of a saturated aqueous solution of ammonium chloride.Extraction by diethyl ether affords a crude product, which can bepurified by silica gel chromatography to give 518-14.

A solution of 518-14 in THF is added dropwise to a solution of n-BuLi(1.2 eq) in THF cooled at −78° C. under a nitrogen atmosphere. Thesolution is stirred for 1 h at −78° C. Excess of HMPA (1.4 eq) is added.After 10 min, a solution of MeI (5 eq) in THF is added. After another 5h at −78° C., 20% aqueous NaH₂PO₄ is added, and the mixture is warmed toroom temperature. Extraction with diethyl ether gives a crude product,which is purified by silica gel chromatography to give 518-15.

Dichlorodicyanoquinone (DDQ) is added to a mixture of compound 518-15 indichloromethane and water. After stirring at room temperature for 2 h.The mixture is extracted with dichloromethane to give a crude product,which is purified by silica gel chromatography to give 518-16.

To a solution of 518-16 in dioxane, is added triphenylphosphine (2 eq.),2-amino-6-chloropurine (2 eq) at room temperature. Diisopropylazodicarboxylate (2 eq, DIAD) is added dropwise via syringe. The mixtureis stirred at room temperature for another 3 h. Water is added to quenchthe reaction. Extraction with ethyl acetate gives a crude product, whichis purified by silica gel chromatography to give 518-17.

Alternatively, a nucleobase may be added by the methods described inCrimmins, M. T. (1998) Tetrahedron 54:9229-9272, such as palladiumcoupling to a cyclopentyl acetate.

To a solution of compound 518-17 in THF is added a 1 M solution oftetrabutylammonium fluoride (1.2 eq, TBAF) at room temperature. Afteranother few hours, a saturated solution of ammonium chloride is added.Extraction with ethyl acetate gives a crude product, which is purifiedby silica gel chromatography to give 518-18.

Compound 18, diethyl bromomethylphosphonate (1.5 eq), and lithiumt-butoxide (1.5 eq) are added to DMF sequentially. The mixture isstirred at 80° C. for several hours. After the mixture is cooled to roomtemperature, a 1 M solution of KH₂PO₄ is added. Extraction with ethylacetate gives a crude product, which is purified by silica gelchromatography to give 518-19.

To a solution of 518-19 in acetone, is added N-methylmorpholine N-oxide(2 eq) and osmium tetraoxide (0.2 eq). The mixture is stirred at roomtemperature for 16 h. A 1 M aqueous solution of sodium sulfite is added.After stirring at room temperature for another hour, the mixture isevaporated to remove most of acetone. The aqueous residue is frozen andlyophilized to give a crude product, which is purified by reversed phaseHPLC to give 518-20.

Iodotrimethylsilane (8 eq, TMS-I) is added to a mixture of 518-20,2,6-lutidine (8 eq) and acetonitrile. After stirring at room temperaturefor 2 h, the mixture is poured onto ice. The mixture is then frozen andlyophilized to give a residue, which is purified by reversed phase HPLCto give 518-21.

518-21 is dissolved in 4 N aqueous NaOH and refluxed for several hours.The mixture is cooled to room temperature, neutralized with 4 N HCl, andpurified with reversed phase HPLC to give 518-22.

Compound 518-22 can be converted to the correspondingdiphosphophosphonate 518-23, and prodrugs using known procedures.

3-Cyclopenten-1-ol 518-24(108 uL, 1.2 mmol, 1.2 eq) is dissolved in 5 mLof dry THF (Scheme 518-8). The solution is cooled to 0° C. A 1.35 Msolution of n-BuLi (0.89 mL, 1.2 mmol, 1.2 eq) is added via syringe.After 10 min, diisopropylphosphonomethyl p-toluenesulfonate (350 mg, 1.0mmol, 1.0 eq) is added. The mixture is stirred in a 45° C. bath for 3.5h. The reaction is quenched with a pH 7 aqueous phosphate buffer.Extraction with diethyl ether gave a crude product, which is purified bysilica gel chromatography (eluted with 45% ethyl acetate in hexane) togive 178 mg of 518-25 (68%).

To a solution of 518-25 (168 mg, 0.69 mmol, 1 eq) in 12 mL of acetone,is added 273 mg of NaHCO₃ in 8 mL of water. The mixture is then cooledto 0° C. Oxone (519 mg, 0.85 mmol, 1.3 eq) in 4 mL of water is addedover 5 min in portions. The mixture is stirred vigorously for 2.5 h. Themixture is then evaporated in vacuo to remove most of the acetone. Theaqueous residue is extracted with ethyl acetate to give a crude product,which is purified by silica gel chromatography to give 518-26 as a clearoil.

To a solution of 518-26 (21 mg, 0.076 mmol, 1.0 eq) in 0.25 mL of DMF,is added cytosine (13 mg, 1.5 eq) and cesium carbonate (6 mg, 0.25 eq)and magnesium t-butoxide. The mixture is heated to 140° C. for severalhours. After cooling to room temperature, the reaction mixture ispurified by reversed phase HPLC to give 12.5 mg of 518-27 (42%). ¹H NMR(CDCl₃): δ 9.60 (br s, 1H), 8.96 (br s, 1H), 7.87 (d, 1H), 6.21 (d, 1H),4.84 (m, 1H), 4.78 (m, 2H), 4.43 (m, 1H), 4.08 (s, 1H), 3.72 (m, 2H),2.82 (m, 1H), 2.33 (m, 1H), 1.83 (m, 2H), 1.38 (m, 12H) ppm.

Alternatively, the methods in WO 03/105770 can be applied to add anucleobase with a nucleophilic amine to a cyclopentyl epoxide.

The conversion from 518-27 to 518-28 is described in Scheme 518-2 above.The conversion of 518-28 to the corresponding diphosphophosphonate518-29 and phosphorus prodrugs, e.g. 518-30 can be accomplished usingprocedures described herein.

Cyclopentyl intermediate 518-31 may be prepared by procedures analogousto those described in U.S. Pat. No. 5,206,244 and U.S. Pat. No.5,340,816 (Scheme 518-4). Diol 518-31 is converted to cyclopentenone518-32 and treated with IBr in the presence of the appropriatephosphonate alcohol to give 518-33. Iodide 518-33 is displaced withinversion to give cyclopentanone intermediate 518-34. Nystedmethylenation (U.S. Pat. No. 3,865,848; Aldrichim. Acta (1993) 26:14)provides exocyclic methylene 518-35, which may be deprotected to give518-36.

Cyclopentanone 518-34 may be a versatile intermediate to form othercompounds of the invention by reduction to cyclopentyl 518-37, or Wittigor Grubb olefination to alkenyl 518-38.

Scheme 518-10 shows intermediate 518-39 is converted to guanosylcyclopentenone 518-40 (J. Am. Chem. Soc. (1972) 94:3213), then treatedwith IBr and diethyl phosphomethanol to furnish iodide 518-41 (J. Org.Chem. (1991) 56:2642) Nucleophilic substitution with AgOAc affordsacetate 518-42. After methylenation using the procedure of Nysted (U.S.Pat. No. 3,865,848; Aldrichim. Acta 1993, 26, 14), to give 518-43, theacetate group is removed by the addition of sodium methoxide and theresulting alcohol is inverted by the Mitsunobo protocol, and a secondacetate deprotection produces 518-44. Desilylation withtetra-butylammonium fluoride (TBAF) of 518-44 will yield 518-45.

Specific Embodiments

Example 221 Synthesis of Exemplary Compounds of the Invention

Synthesis of 519-1: To a solution of 3′-deoxyuridine (995 mg, 4.36 mmol)in 8 mL of anhydrous pyridine was added t-butyldiphenylsilyl chloride(TBDPS-Cl, 1.38 g, 5.01 mmol), and 4-dimethylaminopyridine (DMAP, 27 mg,0.22 mmol). The mixture was stirred at 23 C for 14 h and then cooled to0 C in a ice-water bath. To this mixture was added benzoyl chloride (735mg, 0.61 mL, 5.2 mmol). The mixture was warmed to 23° C. and stirred foranother 2 h. The mixture was concentrated in vacuo to give a paste,which was partitioned between water and ethyl acetate. The aqueous laterwas extracted once with ethyl acetate. The combined ethyl acetate layerwas washed sequentially with 1 M aqueous citric acid, saturated sodiumbicarbonate, and brine. It was dried over anhydrous sodium sulfate andconcentrated in vacuo to give a crude product as a yellow oil.Purification by silica gel chromatography (15-65% ethyl acetate inhexane) gave a colorless oil. Yield 1.35 g (54%). ¹H NMR (DMSO-d6): δ11.38 (s, 1H), 8.01 (d, J=7.9 Hz, 2H), 7.77 (d, J=8.2 Hz, 1H), 7.70-7.40(m, 13H), 5.99 (s, 1H), 5.58 (m, 1H), 7.34 (d, J=8.2 Hz, 1H), 4.47 (m,1H), 4.03 (m, 1H), 3.84 (m, 1H), 2.43 (m, 1H), 2.21 (m, 1H), 1.03 (s,9H) ppm. MS (m/z) 571.1 (M+H⁺), 593.3 (M+Na⁺).

Example 222 Synthesis of Exemplary Compounds of the Invention

Synthesis of 520-2: To a solution of 519-1 (1.31 g, 2.3 mmol) in 5 mL ofanhydrous N,N-dimethylformamide was added benzyl chloromethyl ether(0.54 g, 3.45 mmol), N,N-diisopropylethylamine (446 mg, 0.60 mL, 3.45mmol). The mixture was stirred at 23° C. for 4 h. Water was added. Themixture was extracted with ethyl acetate. The organic layer was washedsequentially with 1 M aqueous citric acid, saturated sodium bicarbonate,and brine. It was dried over anhydrous sodium sulfate and concentratedin vacuo to give a crude product as a yellow oil, which was used in thenext step without further purification.

The crude product obtained above was dissolved in 9 mL of THF. Thesolution was cooled to 0 C. A 1 M solution of TBAF (4.6 mL, 4.6 mmol)was added via syringe. The mixture was warmed to 23° C. and stirred foranother 2 h. An additional 2.3 mL of 1 M TBAF was added. The mixture wasstirred for another 2 h at 23 C. Saturated aqueous ammonium chloride wasadded to the solution. The mixture was evaporated in vacuo to removemost of THF. The aqueous phase was extracted with ethyl acetate. Theaqueous layer was washed with brine. It was then dried over anhydroussodium sulfate and concentrated in vacuo to give a crude product as ayellow oil. Purification by silica gel chromatography (30-80% ethylacetate in hexane) gave a white solid. Yield of 520-2: 805 mg (77% fortwo steps). ¹H NMR (DMSO-d6): δ 8.04 (m, 3H), 7.67 (t, J=7.3 Hz, 1H),7.55 (t, J=7.6 Hz, 2H), 7.30 (m, 5H), 5.98 (s, 1H), 5.78 (d, J=7.9 Hz,1H), 5.55 (m, 1H), 5.31 (s, 2H), 5.22 (m, 1H), 4.57 (s, 2H), 4.41 (m,1H), 3.80 (m, 1H), 3.60 (m, 1H), 2.31 (m, 1H), 2.15 (m, 1H) ppm. MS(m/z) 453.1 (M+H⁺), 475.3 (M+Na⁺).

Example 223 Synthesis of Exemplary Compounds of the Invention

Synthesis of 521-3: To a solution of 520-2 (800 mg, 1.77 mmol) in 3.5 mLof a 1:1 mixture of acetonitrile/water was added iodobenzene diacetate(1.25 g, 3.89 mmol), and TEMPO (55 mg, 0.35 mmol). The mixture wasstirred at 23° C. for 14 h. The mixture was then froze in a −78° C. bathand lyophilized to give a solid residue. This residue was purified bysilica gel chromatography (0-15% methanol in dichloromethane). Product521-3 was obtained as a white solid. Yield: 735 mg (89%). ¹H NMR(DMSO-d6): δ 8.13 (d, J=7.6 Hz, 1H), 8.03 (d, J=7.7 Hz, 2H), 7.68 (m,1H), 7.58 (t, J=7.0 Hz, 2H), 7.29 (m, 5H), 6.04 (s, 1H), 5.85 (d, J=8.3Hz, 1H), 5.62 (m, 1H), 5.31 (s, 2H), 4.87 (m, 1H), 4.58 (s, 2H),2.40-2.20 (m, 2H) ppm. MS (m/z) 467.1 (M+H⁺), 489.3 (M+Na⁺).

Example 224 Synthesis of Exemplary Compounds of the Invention

Synthesis of 522-4: To a deoxygenated solution of 521-3 (730 mg, 1.57mmol) and pyridine (0.51 mL, 6.26 mmol) in 7 mL of anhydrous DMF, wasadded lead tetraacetate (3.47 g, 7.83 mmol). The mixture was stirred at23° C. for 14 h shielded from light. The mixture was diluted with 15 mLof ethyl acetate and 10 mL of water. This mixture filtered through a padof Celite and separated. The aqueous phase was extracted with another 10mL of ethyl acetate. The combined ethyl acetate extract was washed withbrine, dried over sodium sulfate, and evaporated in vacuo to give thecrude product as an oil. The crude product 522-4 was purified by silicagel chromatography (10-50% ethyl acetate in hexane). Products of twodiastereomers were obtained as a white foam. Yield: 400 mg (53%). ¹H NMR(DMSO-d6): δ 8.01 (m, 2H), 7.82-7.63 (m, 2H), 7.57 (m, 2H), 7.31 (m,5H), 6.58 (m, 1H), 6.17 (m, 1H), 5.83 (m, 1H), 5.65 (m, 1H), 5.31 (s,2H), 4.59 (s, 2H), 2.76 and 2.28 (m, 1H), 2.10 (m, 1H), 2.07 (s, 3H)ppm. MS (m/z) 481.0 (M+H⁺), 503.3 (M+Na⁺).

Example 225 Synthesis of Exemplary Compounds of the Invention

Synthesis of 523-5a: To a solution of 522-4 (300 mg, 0.63 mmol) in 6 mLof anhydrous dichloromethane was added diethyl hydroxymethylphosphonate(0.37 mL, 2.5 mmol), followed by trimethylsilyltrifluoromethanesulfonate (0.34 mL, 1.88 mmol). The mixture was stirredat 23° C. for 6 h. Triethylamine (0.44 mL, 3.15 mmol) was added,followed by water. The mixture was extracted with ethyl acetate. Theorganic layer was washed with 1 M aqueous citric acid, saturated sodiumbicarbonate, and brine. It was then dried over anhydrous sodium sulfate,and evaporated in vacuo to give a residue. This crude product waspurified by silica gel chromatography (75-95% ethyl acetate in hexane)to give two products, which were diastereomers of each other shown above(523-5a and 523-5b). Yield of 523-5a: 53 mg (14%). Yield of 523-5b: 129mg (35%).

Analytical data for 5a: ¹H NMR (Acetonitrile-d3): δ 8.04 (d, J=7.0 Hz,2H), 7.77 (d, J=7.9 Hz, 1H), 7.69 (t, J=7.5 Hz, 1H), 7.53 (m, 2H), 7.33(m, 5H), 6.38 (d, J=4.0 Hz, 1H), 5.80 (d, J=8.2 Hz, 1H), 5.63 (m, 1H),5.52 (m, 1H), 5.41 (s, 2H), 4.64 (s, 2H), 4.17 (m, 4H), 4.08 (dd,J=13.8, 10.1 Hz, 1H), 3.92 (dd, J=13.7, 9.5 Hz, 1H), 2.66-2.42 (m, 2H),1.35 (t, J=7.0 Hz, 6H) ppm. MS (m/z) 589.2 (M+H⁺), 611.3 (M+Na⁺).Stereochemistry of 523-5a was confirmed by additional 2D NMRexperiments.

Analytical data for 523-5b: ¹H NMR (Acetonitrile-d3): δ 8.08 (d, J=7.3Hz, 2H), 7.69 (t, J=7.5 Hz, 1H), 7.55 (m, 2H), 7.43 (d, J=8.2 Hz, 1H),7.36 (m, 5H), 6.11 (d, J=2.4 Hz, 1H), 5.77 (d, J=8.3 Hz, 1H), 5.57 (m,2H), 5.41 (s, 2H), 4.66 (s, 2H), 4.12 (m, 5H), 3.88 (dd, J=14.0, 5.2 Hz,1H), 2.82 (m, 1H), 2.25 (m, 1H), 1.27 (t, J=7.0 Hz, 6H) ppm. MS (m/z)589.0 (M+H⁺), 611.2 (M+Na⁺).

Example 226 Synthesis of Exemplary Compounds of the Invention

Synthesis of 524-6: To a solution of 523-5a (110 mg, 0.19 mmol) in 3 mLof acetonitrile was added 2,6-lutidine (0.43 mL, 3.74 mmol), followed byiodotrimethylsilane (0.53 mL, 3.74 mmol). After stirring at 23 C for 30min, the mixture was heated to 40° C. and stirred at that temperaturefor another 4 h. The reaction mixture was cooled to 23° C. Triethylamine(0.52 mL, 3.74 mmol) was added, followed by water (10 mL). The aqueousmixture was extracted twice with 5 mL of diethyl ether. The resultingaqueous solution was frozen in a −78° C. bath and was lyophilized togive a yellow solid. This crude product was purified by reversed phaseHPLC to give 524-6 as a light yellow solid. Yield 26 mg (34%). MS (m/z)411.3 (M−H—).

Example 227 Synthesis of Exemplary Compounds of the Invention

Synthesis of 525-7: Phosphonate 524-6 (12 mg, 0.029 mmol),carbonyldiimidazole (47 mg, 0.29 mmol), and tri-n-butylamine (5.4 mg,0.029 mmol) were dissolved in 0.3 mL of anhydrous dimethylformamide(DMF). The mixture was stirred at 23° C. for 4 h. MeOH (0.020 mL) wasadded and the mixture was stirred for another 30 min. A solution oftributylammonium pyrophosphate (159 mg, 0.29 mmol) in 0.63 mL ofanhydrous DMF was added. The resulting mixture was stirred at 23° C. for14 h. The mixture was evaporated in vacuo to remove most of the DMF. Theresidue was dissolved in 5 mL of water and was purified by ion-exchangechromatography (DEAE-cellulose resin, 0-50% triethylammonium bicarbonatein water) to give a white solid, which was used directly in the nextreaction.

The product obtained above was dissolved in 2 mL of water. A 0.3 mL of a1 M solution of sodium hydroxide in water was added. The mixture wasstirred at 23° C. for 40 min. Acetic acid was added to adjust the pH ofthe solution to 5. The solution was diluted with water and purified withan ion-exchange column (DEAE-cellulose resin, 0-50% triethylammoniumbicarbonate in water) to give diphosphophosphonate 525-7 as a whitesolid, which is the triethylammonium salt of the structure shown above.Yield 10 mg (45% for two steps). ¹H NMR (D₂O): δ 7.79 (d, J=7.6 Hz, 1H),5.89 (m, 1H), 5.85 (d, J=7.6 Hz, 1H), 5.41 (m, 1H), 4.49 (m, 1H),4.02-3.65 (m, 2H), 3.06 (m, 18H), 2.20 (m, 2H), 1.14 (m, 27H) ppm. ³¹PNMR (D₂O): δ 7.46 (d, 1P), −9.45 (d, 1P), −23.11 (t, 1P) ppm. MS (m/z)467.0 (M−H⁻).

Example 228 Synthesis of Exemplary Compounds of the Invention

Synthesis of 526-8: To a solution of 524-6 (16 mg, 0.039 mmol) in 0.4 mLof water was added NaOH (7.8 mg, 0.19 mmol). The solution was stirred at23° C. for 1 h. Acetic acid (0.012 mL) was added to the solution. Themixture was then purified by reversed phase HPLC (eluted with 100%water) to give 4.6 mg of 526-8 as a white solid (38% yield). ¹H NMR(D₂O): δ 7.83 (d, J=8.3 Hz, 1H), 5.86 (d, J=3.4 Hz, 1H), 5.82 (d, J=7.9Hz, 1H), 4.48 (m, 1H), 3.68 (m, 1H), 3.37 (m, 1H), 2.16 (m, 2H) ppm. ³¹PNMR (D₂O): δ 12.60 (s, 1P) ppm. MS (m/z) 615.1 (2M−H⁻).

A solution of tert-butyl hydroperoxide (t-BuOOH) in benzene (68%, 3 eq)is added dropwise to a solution of allylic alcohol 526-1 (synthesized asdescribed in Tet. Lett., 38: 2355 (1997)) and VO(acac)₂ in benzene(final concentration 0.1 M) at room temperature (Scheme 526-1). After 1h of stirring at room temperature, saturated aqueous Na₂S₂O₃ is added tothe reaction mixture. The resulting solution is extracted with EtOAc,washed with H₂O, and dried over sodium sulfate. After removal ofsolvent, the crude product 526-2 is purified by column chromatography onsilica.

Epoxide 526-2 and p-anisylchlorodiphenylmethane (1.5 eq) is dissolved inanhydrous pyridine (0.17 M) and stirred at 25° C. for 2d. Solvents wereremoved under reduced pressure and the residue dissolved in EtOAc. Theorganics were washed with water, saturated aqueous NaHCO₃, and driedover sodium sulfate. After removal of solvent, the crude product 526-3is purified by column chromatography on silica.

To a solution of methyltriphenylphosphonium bromide (2 eq) in anhydrousTHF at −78° C. is added n-butyllithium (2.2 eq). The solution is allowedto warm to room temperature and stirred for 20 min. After recooling to−78° C., this solution is added to fully protected epoxide 526-3 in THF(final concentration 0.06 M). The reaction mixture is allowed to warm toroom temperature and stirred for 12 h at which point H₂O is added andextracted with diethyl ether. The combined organics were dried oversodium sulfate. After removal of solvent, the crude product 526-4 ispurified by column chromatography on silica.

Sodium hydride (1 eq) and 2-amino-4-chloro-7H pyrrolo[2,3-d]pyrimidine(1 eq) were dissolved in anhydrous DMF (0.06 M) and stirred at 120° C.for 10 min. A solution of 526-4 in DMF is then added and the reactionmixture is stirred 12 h at 120° C. at which point the solvents wereevaporated under reduced pressure. The residue is dissolved in CH₂Cl₂,washed with H₂O, and dried over sodium sulfate. After removal ofsolvent, the crude product 526-5 is purified by column chromatography onsilica.

Compound 526-5 is dissolved in dichloromethane and added to a solutionof 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one(Aldrich, Dess-Martin periodinane, 4 eq) in dichloromethane (finalconcentration 0.06 M). The reaction mixture is stirred at roomtemperature for 4 d at which point it is diluted with EtOAc and pouredinto a solution of sodium thiosulfate in saturated aqueous sodiumbicarbonate solution. The organic layer is separated and dried oversodium sulfate. After removal of solvent, the crude product 6 ispurified by column chromatography on silica.

A solution of ketone 526-6 in anhydrous THF is added to a solution ofmethylmagnesium bromide (4 eq) in anhydrous THF (0.1 M) at −78° C. Thereaction mixture is stirred for 12 h at 60° C. at which point thereaction is quenched with saturated aqueous NH₄Cl solution. The mixtureis filtered over celite and washed with EtOAc. The combined organicswere washed with saturated aqueous NH₄Cl, water and dried over sodiumsulfate. After removal of solvent, the crude product 526-7 is purifiedby column chromatography on silica.

A solution of alcohol 7 in anhydrous THF (0.06 M) is treated with asolution of tetrabutylammonium fluoride (1.5 eq) in THF at roomtemperature. The reaction mixture is stirred for 3 h at which point thesolvents were evaporated. The crude desilylated diol 526-8 is purifiedby column chromatography on silica.

To a solution of diol 526-8 and benzenesulfonic aciddiisopropoxy-phosphorylmethyl ester (1.2 eq) in anhydrous DMF (0.1 M) isadded magnesium tert-butoxide (1 eq). The reaction mixture is heated to80° C. for 12 h. After cooling to room temperature, 1 N citric acid isadded and extracted with EtOAc. The organics were neutralized withsaturated aqueous NaHCO₃, washed with saturated aqueous NaCl, and driedover sodium sulfate. After removal of solvent, the crude product 526-9is purified by column chromatography on silica.

Compound 526-9 is dissolved in 80% acetic acid and stirred 12 h at roomtemperature. After removal of solvent, the crude product 526-10 ispurified by column chromatography on silica.

Phosphonate ester 526-10 and 2,6-lutidine (8 eq) is dissolved in CH₃CNand treated with trimethylsilyliodide (8 eq). After stirring for 3 h atroom temperature, triethylamine (8 eq) is added followed by methanol.After removal of solvent, the crude product 526-11 is purified by columnchromatography on silica.

Phosphonic diacid 526-11 is dissolved in 1,4-dioxane and treated with 4N NaOH and heated to 100° C. for 4 h. After cooling to room temperature,the reaction mixture is neutralized with 4N HCl. After removal ofsolvent, the crude product is purified by column chromatography onsilica to provide 526-12.

Compound 526-13 (Paquette et al in J. Org. Chem. (1997) 62:1730-1736) istreated with p-methoxybenzyl bromide (1.5 eq.), sodium hydride (1.4 eq)in dry DMF at room temperature (Scheme 526-2). The reaction is monitorby TLC for the disappearance of 526-13. The reaction is quenched by theaddition of a saturated aqueous solution of ammonium chloride.Extraction by diethyl ether affords a crude product, which can bepurified by silica gel chromatography to give 526-14.

A solution of 526-14 in THF is added dropwise to a solution of n-BuLi(1.2 eq) in THF cooled at −78° C. under a nitrogen atmosphere. Thesolution is stirred for 1 h at −78° C. Excess of HMPA (1.4 eq) is added.After 10 min, a solution of MeI (5 eq) in THF is added. After another 5h at −78° C., 20% aqueous NaH₂PO₄ is added, and the mixture is warmed toroom temperature. Extraction with diethyl ether gives a crude product,which is purified by silica gel chromatography to give 526-15.

Dichlorodicyanoquinone (DDQ) is added to a mixture of compound 526-15 indichloromethane and water. After stirring at room temperature for 2 h.The mixture is extracted with dichloromethane to give a crude product,which is purified by silica gel chromatography to give 526-16.

To a solution of 526-16 in dioxane, is added triphenylphosphine (2 eq.),2-amino-6-chloropurine (2 eq) at room temperature. Diisopropylazodicarboxylate (2 eq, DIAD) is added dropwise via syringe. The mixtureis stirred at room temperature for another 3 h. Water is added to quenchthe reaction. Extraction with ethyl acetate gives a crude product, whichis purified by silica gel chromatography to give 526-17.

To a solution of compound 526-17 in THF is added a 1 M solution oftetrabutylammonium fluoride (1.2 eq, TBAF) at room temperature. Afteranother few hours, a saturated solution of ammonium chloride is added.Extraction with ethyl acetate gives a crude product, which is purifiedby silica gel chromatography to give 526-18.

Compound 526-18, diethyl bromomethylphosphonate (1.5 eq), and lithiumt-butoxide (1.5 eq) are added to DMF sequentially. The mixture isstirred at 80° C. for several hours. After the mixture is cooled to roomtemperature, a 1 M solution of KH₂PO₄ is added. Extraction with ethylacetate gives a crude product, which is purified by silica gelchromatography to give 526-19.

To a solution of 526-19 in acetone, is added N-methylmorpholine N-oxide(2 eq) and osmium tetraoxide (0.2 eq). The mixture is stirred at roomtemperature for 16 h. A 1 M aqueous solution of sodium sulfite is added.After stirring at room temperature for another hour, the mixture isevaporated to remove most of acetone. The aqueous residue is frozen andlyophilized to give a crude product, which is purified by reversed phaseHPLC to give 526-20.

Iodotrimethylsilane (8 eq, TMS-I) is added to a mixture of 526-20,2,6-lutidine (8 eq) and acetonitrile. After stirring at room temperaturefor 2 h, the mixture is poured onto ice. The mixture is then frozen andlyophilized to give a residue, which is purified by reversed phase HPLCto give 526-21.

526-21 is dissolved in 4 N aqueous NaOH and refluxed for several hours.The mixture is cooled to room temperature, neutralized with 4 N HCl, andpurified with reversed phase HPLC to give 526-22.

Compound 526-22 can be converted to the correspondingdiphosphophosphonate 526-23, and prodrugs using known procedures.

3-Cyclopenten-1-ol 526-24 (108 uL, 1.2 mmol, 1.2 eq) is dissolved in 5mL of dry THF The solution is cooled to 0° C. A 1.35 M solution ofn-BuLi (0.89 mL, 1.2 mmol, 1.2 eq) is added via syringe. After 10 min,diisopropylphosphonomethyl p-toluenesulfonate (350 mg, 1.0 mmol, 1.0 eq)is added. The mixture is stirred in a 45° C. bath for 3.5 h. Thereaction is quenched with a pH 7 aqueous phosphate buffer. Extractionwith diethyl ether gave a crude product, which is purified by silica gelchromatography (eluted with 45% ethyl acetate in hexane) to give 178 mgof 526-25 (68%).

To a solution of 526-25 (168 mg, 0.69 mmol, 1 eq) in 12 mL of acetone,is added 273 mg of NaHCO₃ in 8 mL of water. The mixture is then cooledto 0° C. Oxone (519 mg, 0.85 mmol, 1.3 eq) in 4 mL of water is addedover 5 min in portions. The mixture is stirred vigorously for 2.5 h. Themixture is then evaporated in vacuo to remove most of the acetone. Theaqueous residue is extracted with ethyl acetate to give a crude product,which is purified by silica gel chromatography to give 526-26 as a clearoil.

To a solution of 526-26 (21 mg, 0.076 mmol, 1.0 eq) in 0.25 mL of DMF,is added cytosine (13 mg, 1.5 eq) and cesium carbonate (6 mg, 0.25 eq)and magnesium t-butoxide. The mixture is heated to 140° C. for severalhours. After cooling to room temperature, the reaction mixture ispurified by reversed phase HPLC to give 12.5 mg of 526-27 (42%). ¹H NMR(CDCl₃): δ 9.60 (br s, 1H), 8.96 (br s, 1H), 7.87 (d, 1H), 6.21 (d, 1H),4.84 (m, 1H), 4.78 (m, 2H), 4.43 (m, 1H), 4.08 (s, 1H), 3.72 (m, 2H),2.82 (m, 1H), 2.33 (m, 1H), 1.83 (m, 2H), 1.38 (m, 12H) ppm.

The conversion from 526-27 to 526-28 is described in Scheme 526-2 above.The conversion of 526-28 to the corresponding diphosphophosphonate526-29 and phosphorus prodrugs, e.g. 526-30 can be accomplished usingprocedures described herein.

Cyclopentyl intermediate 526-31 may be prepared by procedures analogousto those described in U.S. Pat. No. 5,206,244 and U.S. Pat. No.5,340,816 (Scheme 526-4). Diol 526-31 is converted to cyclopentenone526-32 and treated with IBr in the presence of the appropriatephosphonate alcohol to give 526-33. Iodide 526-33 is displaced withinversion to give cyclopentanone intermediate 526-34. Nystedmethylenation (U.S. Pat. No. 3,865,848; Aldrichim. Acta (1993) 26:14)provides exocyclic methylene 526-35, which may be deprotected to give526-36.

Cyclopentanone 526-34 may be a versatile intermediate to form othercompounds of the invention by reduction to cyclopentyl 526-37, or Wittigor Grubb olefination to alkenyl 526-38.

Scheme 526-5 shows intermediate 526-39 is converted to guanosylcyclopentenone 526-40 (J. Am. Chem. Soc. (1972) 94:3213), then treatedwith IBr and diethyl phosphomethanol to furnish iodide 526-41 (J. Org.Chem. (1991) 56:2642) Nucleophilic substitution with AgOAc affordsacetate 526-42. After methylenation using the procedure of Nysted (U.S.Pat. No. 3,865,848; Aldrichim. Acta 1993, 26, 14), to give 526-43, theacetate group is removed by the addition of sodium methoxide and theresulting alcohol is inverted by the Mitsunobo protocol, and a secondacetate deprotection produces 526-44. Desilylation withtetra-butylammonium fluoride (TBAF) of 526-44 will yield 526-45.

Synthesis of 2′-C-Me-UP

Example 229 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 527-1: L-Xylose (36.2 g) and anhydrous CuSO₄ wereplaced in a 500 mL round bottomed flask. Acetone (220 mL) was added. Tothis slurry stirred at room temperature was added 3.6 mL of 96% sulfuricacid. The mixture was stirred for another 24 h at room temperature undera nitrogen atmosphere. The mixture is filtered to remove solid material.The solids were washed with 50 mL of acetone. To the combined filtratewas added 25.3 mL of conc. ammonium hydroxide. The precipitates wereremoved by filtration. The filtrate was evaporated in vacuo to give anoil, which was co-evaporated twice with absolute ethanol to give ayellow oil. The above crude product was stirred with 160 mL of 0.06 Maqueous HCl vigorously at room temperature for 2.5 h. The reactionmixture was homogeneous at the end of the reaction. Solid NaHCO₃ (3.26g) was added in portions. After gas evolution had stopped, the mixturewas filtered. The filtrate was frozen and lyophilized overnight to givea syrup, which was dissolved in ethyl acetate and dried over anhydrousNa₂SO₄ to give the desired diol as a yellow oil. Proton NMR showed theproduct to be >95% pure. Yield of this crude product: 44.5 g (96%). ¹HNMR (DMSO-d₆, 300 MHz): δ 5.79 (d, J=3.6 Hz, 1H), 5.13 (d, J=4.9 Hz,1H), 4.61 (t, J=5.6 Hz, 1H), 4.36 (d, J=3.6 Hz, 1H), 4.10-3.91 (m, 2H),3.60 (m, 1H), 3.51 (m, 1H), 1.37 (s, 3H), 1.22 (s, 3H) ppm

Example 230 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 528-2: 1,2-O-isopropylidene-L-xylose (5 g, 26.3mmol, 1.0 eq.) and 2-iodobenzoyl chloride (7.01 g, 26.3 mmol) weredissolved in anhydrous dichloromethane (25 mL). The solution was cooledin an ice-water bath. Triethylamine (3.85 mL, 27.6 mmol, 1.05 eq.) wasadded dropwise via syringe. The mixture was stirred at 0° C. for 30 minand slowly warmed to room temperature over 1 h. Water was added to thereaction mixture. The mixture was washed with 1 M aqueous HCl. Theaqueous wash was extracted with 20 mL of dichloromethane. The combinedorganic extract was washed with a mixture of 20 mL of brine and 5 mL ofsaturated aqueous sodium bicarbonate. The organic layer was dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo to give a brownoil. This crude product was purified by silica gel chromatography(eluted with 0-50% EtOAc in hexane) to give the desired mono-ester as ayellow oil. Yield: 7.6 g (69%). ¹H NMR (DMSO-d₆, 300 MHz): δ 8.02 (d,J=7.3 Hz, 1H), 7.73 (dd, J=7.8, 1.7 Hz, 1H), 7.52 (t, J=7.3 Hz, 1H),7.29 (td, J=7.7, 1.8 Hz, 1H), 5.88 (d, J=3.7 Hz, 1H), 5.51 (m, 1H), 4.45(m, 2H), 4.12 (m, 1H), 1.38 (s, 3H), 1.24 (s, 3H) ppm. MS (m/z):calculated 420.01 (M+H⁺), 443.00 (M+Na⁺), found 420.9 (M+H⁺), 443.0(M+Na⁺).

Example 231 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 529-3: The product obtained in the previous step(7.6 g, 18.1 mmol, 1.0 eq.) was dissolved in 35 mL of anhydrousdichloromethane. Dess-Martin periodinane (9.6 g, 22.6 mmol, 1.25 eq.)was added. The mixture was stirred at room temperature for 14 h. A 1 Msolution of sodium sulfite (7.5 mL) was added. The resulting mixture wasstirred for another 2 h at room temperature. A saturated solution ofNaHCO₃ was added in portions to adjust the pH of the aqueous phase to 6.The two layers were separated. The aqueous phase was extracted twicewith 15 mL of dichloromethane. The combined organic extract was washedwith brine, dried over anhydrous Na₂SO₄ for 4 h with good stirring. Itwas then filtered, and dried over excess amount of anhydrous MgSO₄overnight with good stirring. The mixture was filtered and concentratedin vacuo to give a clear oil as product, which was used without furtherpurification in the subsequent step. Yield: 6.7 g (89%). ¹H NMR(DMSO-d₆, 300 MHz): δ 8.02 (d, J=7.9 Hz, 1H), 7.71 (dd, J=7.8, 1.7 Hz,1H), 7.52 (t, J=7.4 Hz, 1H), 7.29 (td, J=7.6, 1.5 Hz, 1H), 6.16 (d,J=4.6 Hz, 1H), 4.85 (m, 1H), 4.63 (d, J=4.6 Hz, 1H), 4.54 (dd, J=12.2,2.7 Hz, 1H), 4.42 (dd, J=12.2, 4.3 Hz, 1H), 1.41 (s, 3H), 1.34 (s, 3H)ppm. MS (m/z): calculated 458.99 (M+H₂O+Na⁺), found 459.03 (M+H₂O+Na⁺).

Example 232 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 530-4: The product obtained in the previous step(6.15 g, 14.7 mmol, 1.0 eq.) was dissolved in 29 mL of anhydrous THF.The solution was cooled in an ice-water bath. A 3.0 M solution of methylmagnesium bromide in diethyl ether (5.39 mL, 16.2 mmol, 1.1 eq.) wasadded dropwise via syringe. The mixture was stirred at 0° C. for 2 h.Aqueous citric acid solution (1 M, 10 mL) was added to the reactionmixture. The resulting mixture was evaporated in vacuo to remove most ofTHF. The aqueous residue was extracted twice with 10 mL of EtOAc. Theorganic extract was washed with saturated NaHCO₃ and brine. The organicphase was dried over anyhydrous sodium sulfate, filtered, and evaporatedin vacuo to give a white solid as product. Yield: 6.11 g (96%). ¹H NMR(DMSO-d₆, 300 MHz): δ 8.01 (dd, J=8.0, 1.0 Hz, 1H), 7.71 (dd, J=7.8, 1.7Hz, 1H), 7.52 (td, J=7.5, 1.0 Hz, 1H), 7.29 (td, J=7.7, 1.8 Hz, 1H),5.72 (d, J=3.7 Hz, 1H), 5.13 (s, 1H), 4.46 (dd, J=11.6, 2.3 Hz, 1H),4.20 (dd, J=11.7, 8.5 Hz, 1H), 4.12 (d, J=3.6 Hz, 1H), 4.08 (dd, J=8.5,2.1 Hz, 1H), 1.45 (s, 3H), 1.26 (s, 3H), 1.06 (s, 3H) ppm. MS (m/z):calculated 457.01 (M+Na⁺), found 457.27 (M+Na⁺).

Example 233 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 531-5: To a solution of 530-4 (6.1 g, 14.1 mmol)in 20 mL of anhydrous pyridine, was added triethylamine (3.13 mL, 22.5mmol), DMAP (0.343 g, 2.8 mmol), followed by benzoyl chloride (2.61 mL,22.5 mmol). The mixture was stirred at 70 C for 36 h, and then cooled toroom temperature. The mixture was evaporated in vacuo to remove most ofthe pyridine. The residue was acidified with 1 M aqueous citric acid.The resulting mixture was extracted twice with ethyl acetate. Thecombined organic layer was washed with saturated NaHCO₃, and brine,dried over anhydrous Na₂SO₄, and evaporated in vacuo to give a crudeproduct. This crude product was purified by silica gel chromatography(0-35% ethyl acetate in hexane) to give 7.0 g (92%) of 5. ¹H NMR(DMSO-d₆, 300 MHz): δ 8.03 (d, J=8.2 Hz, 1H), 7.90 (d, J=7.5 Hz, 2H),7.79 (d, J=7.7 Hz, 1H), 7.64 (t, J=7.9 Hz, 1H), 7.5 (m, 3H), 7.30 (t,J=7.6 Hz, 1H), 5.92 (d, J 33.7 Hz, 1H), 4.92 (d, J=3.5 Hz, 1H), 4.63 (m,1H), 4.46 (m, 2H), 1.50 (s, 3H), 1.39 (s, 3H), 1.25 (s, 3H) ppm. MS(m/z) 589.2 (M+H⁺), 611.3 (M+Na⁺). MS (m/z): calculated 561.04 (M+Na⁺),found 561.06 (M+Na⁺).

Example 234 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 532-6: To a solution of 531-5 (7.0 g, 13 mmol) in26 mL of glacial acetic acid, was added acetic anhydride (7.7 mL). Thesolution was cooled in an ice-water bath. Concentrated sulfuric acid(1.9 mL) was added dropwise via syringe over 10 min. The cooling bathwas removed and the solution was allowed to warm to room temperature andstirred at that temperature for another 20 h. The reaction mixture waspoured into a mixture of 75 mL of diethyl ether and 75 g of ice. Thelayers were separated and the aqueous layer was extracted with 75 mL ofdiethyl ether. The combined ether extract was stirred with 250 mL ofwater. Solid NaHCO₃ was added in portions until gas evolution hadstopped. The layers were separated. The aqueous layer was extracted with75 mL of ether. The combined ether extract was washed with brine, driedover anhydrous MgSO₄, and concentrated in vacuo to give 6 as a yellowfoam. Two diastereomers of the same molecular weight were present in theproduct mixture, presumably the two anomers. Yield: 6.76 g (89%). Thiscrude product was used without further purification. MS (m/z):calculated 605.03 (M+Na⁺), found 604.93 (M+Na⁺).

Example 235 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 533-7: 532-6 (6.76 g, 11.6 mmol) was dissolved in22 mL of dichloromethane. The solution was cooled in an ice-water bath.A solution of SnCl₄ in dichloromethane (1.0 M, 29 mL, 29 mmol) was addedvia syringe. The cooling bath was removed and the mixture was warmed toroom temperature and stirred for another hour. The mixture was againcooled to 0° C. Triethylamine (15 mL) was added via syringe. Theresulting solution was poured onto a mixture of 75 g of ice and 75 mL ofEtOAc. The mixture was filtered through a pad of Celite. The solids werewashed thoroughly with EtOAc. The combined filtrate was washed withsaturated NaHCO₃, brine, dried over Na₂SO₄, and concentrated in vacuo togive a crude product, which was purified by silica gel chromatography(25-75% EtOAc in hexane) to give 7 as a light yellow foam. Yield: 6.0 g(75%). ¹H NMR (DMSO-d₆, 300 MHz): δ 8.00 (d, J=7.9 Hz, 1H), 7.86 (d,J=8.0 Hz, 2H), 7.79 (d, J=7.9 Hz, 1H), 7.61 (t, J=7.2 Hz, 1H), 7.45 (m,3H), 7.25 (t, J=7.3 Hz, 1H), 5.28 (s, 1H), 5.07 (s, 1H), 4.65 (m, 2H),4.49 (m, 1H), 4.10-3.90 (m, 5H), 3.83 (dd, J=13.9, 9.0 Hz, 1H), 1.88 (s,3H), 1.69 (s, 3H), 1.18 (t, J=6.9 Hz, 6H) ppm. MS (m/z): calculated713.06 (M+Na⁺), found 713.08 (M+Na⁺).

Example 236 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 534-8: To a solution of 533-7 (4.7 g, 6.8 mmol) in30 mL of dichloromethane was added 27.2 mL of a 1.0 M aqueous solutionof KH₂PO₄. A 0.8 M solution of NaOCl in water was added. The mixture wasstirred at room temperature for 1 h. Methanol (10 mL) was added. SolidK2CO3 was added in portions until the pH of the aqueous phase reached9-10. The mixture was stirred for another hour at room temperature. An 1M aqueous solution of Na₂SO₃ (10 mL) was added and the mixture wasstirred for another 30 min at room temperature. The two layers wereseparated. The aqueous layer was further extracted with dichloromethane.The combined organic layer was washed with brine, dried over anhydrousNa2SO4, and evaporated in vacuo to give 534-8 as a yellow foam, whichwas used directly in the next step without further purification. ¹H NMR(DMSO-d₆, 300 MHz): δ 7.94 (d, J=7.8 Hz, 2H), 7.68 (t, J=7.5 Hz, 1H),7.54 (t, J=7.6 Hz, 2H), 5.28 (d, J=1.0 Hz, 1H), 5.05 (d, J=1.2 Hz, 1H),4.98 (t, J=5.6 Hz, 1H), 4.34 (dd, J=6.5, 4.6 Hz, 1H), 4.11-3.95 (m, 5H),3.86 (dd, J=13.7, 8.8 Hz, 1H), 3.76 (m, 1H), 3.63 (m, 1H), 1.93 (s, 3H),1.64 (s, 3H), 1.25 (t, J=7.0 Hz, 6H) ppm.

³¹P NMR (DMSO-d₆): δ 20.63 (s, 1P) ppm. MS (m/z): calculated 483.14(M+Na⁺), found 483.30 (M+Na⁺).

Example 237 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 535-9: To a mixture of 533-8 obtained above and6.8 mL of acetonitrile and 6.8 mL of water, was added iodobenzenediacetate (4.97 g, 15 mmol), and TEMPO (0.213 g, 1.36 mmol). The mixturewas stirred vigorously for 6 h at room temperature. It was then frozenand lyophilized to give a orange colored solid, which was dissolved indichloromethane and purified by silica gel chromatography (0-10% MeOH inCH2Cl2) to give 534-9 as a light yellow solid. Yield: 2.8 g (87% for twosteps). ¹H NMR (DMSO-d₆, 300 MHz): δ 13.39 (br s, 1H), 7.97 (d, J=7.8Hz, 2H), 7.70 (t, J=7.3 Hz, 1H), 7.56 (t, J=7.5 Hz, 2H), 5.35 (s, 1H),5.16 (s, 1H), 4.89 (s, 1H), 4.18 (dd, J=13.7, 8.8 Hz, 1H), 4.06 (m, 4H),3.88 (dd, J=13.4, 9.7 Hz, 1H), 1.86 (s, 3H), 1.69 (s, 3H), 1.24 (dt,J=7.0, 2.7 Hz, 6H) ppm. ³¹P NMR (DMSO-d₆): δ 20.79 (s, 1P) ppm. MS(m/z): calculated 473.12 (M−H⁻), found 472.95 (M−H—).

Example 238 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 536-10: To a solution of 535-9 (474 mg, 1.0 mmol)in 2.0 mL of anhydrous DMF, was added pyridine (238 mg, 3.0 mmol), andlead tetraacetate (1.33 g, 3.0 mmol). The mixture was stirred whileshielded from light for 7 h at room temperature. It was then poured intoa mixture of 10 g of ice and 10 mL of diethyl ether. The mixture wasfiltered to remove precipitates. The two layers of the filtrate wereseparated. The aqueous phase was extracted twice with ether. Thecombined ether extract was washed with 1 M citric acid, saturatedNaHCO₃, and brine. After drying with anhydrous MgSO4, the ether solutionwas concentrated in vacuo to give crude 536-10 as a colorless oil, whichwas used without further purification. Yield: 255 mg (52%). MS (m/z):calculated 511.13 (M+Na⁺), found 511.11 (M+Na⁺).

Example 239 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 537-11: To a solution of 536-10 (211 mg, 0.43mmol) in 2.0 mL of anhydrous acetonitrile, was addedO,O-bis(trimethylsilyl)uracil (443 mg, 1.73 mmol), and TMS-OTf (384 mg,1.73 mmol). The mixture was stirred at room temperature for 3 h. Anadditional 443 mg of O,O-bis(trimethylsilyl)uracil was added, and themixture was stirred for another 4 h at room temperature. 2,6-Lutidine(371 mg, 3.46 mmol) was added dropwise via syringe, followed by TMS-I(259 mg, 1.3 mmol). The mixture was stirred for another hour at roomtemperature and then poured onto 10 g of ice. The mixture frozen andfiltered through a pad of Celite. The filtrate was frozen andlyophilized to give a yellow solid, which was dissolved in water andpurified by reversed phase HPLC to give 537-11 as a white solid. Yield:20 mg (10%). MS (m/z): calculated 483.08 (M−H⁻), found 483.34 (M−H⁻).

Example 240 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 538-12: To a solution of 537-11 (17 mg, 0.035mmol) in 0.3 mL water, was added NaOH (4.3 mg, 0.11 mmol). Afterstirring at room temperature for 2 h, the mixture acidified withtrifluoroacetic acid and purified by HPLC to give 538-12 as a whitepowder. Yield: 5 mg (42%). ¹H NMR (D₂O, 300 MHz): δ 7.74 (d, J=8.2 Hz,1H), 5.97 (s, 1H), 5.78 (d, J=8.2 Hz, 1H), 5.10 (d, J=4.9 Hz, 1H), 3.86(dd, J=12.9, 10.0 Hz, 1H), 3.78 (d, J=4.7 Hz, 1H), 3.65 (dd, I=12.7, 9.3Hz, 1H), 1.10 (s, 3H) ppm. ³¹P NMR (D₂O): δ 14.60 (s, 1P) ppm. MS (m/z):calculated 337.04 (M−H⁻), found 337.38 (M−H⁻).

Example 241 Synthesis of Exemplary Compounds of the Invention

Synthesis of 3′-Deoxy-CP

Example 242 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 540-2 To a stirred solution of 527-1 (50 mg, 0.11mmol) in 1 mL of acetonitrile under nitrogen was added2,4,6-triisopropylbenzenesulfonyl chloride (65 mg, 0.21 mmol), DMAP (26mg, 0.21 mmol), and triethylamine (22 mg, 0.21 mmol). The mixture wasstirred at room temperature for 4 h. Aqueous ammonia (29%, 1 mL) wasadded. The mixture was stirred at room temperature for 2 h. Extractionwith EtOAc, followed by purification by silica gel chromatography gave540-2 as a white solid. MS (m/z): calculated 468.15 (M+H⁺), found 468.0(M+H⁺).

Example 243 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 541-3: To a solution of 540-2 obtained above inacetonitrile was added 2,6-lutidine (118 mg, 1.10 mmol) and TMS-I (165mg, 0.84 mmol). The mixture was stirred at room temperature for 2 h.Triethylamine was added, followed by water. The mixture was then frozenand lyophilized to give a solid residue. This crude product was purifiedby reversed phase HPLC to give 541-3 as a white solid. Yield 44 mg (97%for two steps). MS (m/z): calculated 410.1 (M−H⁻), found 410.2 (M−H⁻).

Example 244 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 542-4: To solution of 541-3 (40 mg, 0.097 mmol) in0.5 mL of water was added NaOH (20 mg, 0.5 mmol). The solution wasstirred at room temperature for 30 min. Reversed phase HPLC purificationgave 542-4 as a white solid. Yield: 28 mg (94%). ¹H NMR (D2O, 300 MHz) δ7.79 (d, J=7.6 Hz, 1H), 5.95 (d, J=7.6 Hz, 1H), 5.88 (d, J=2.8 Hz, 1H),5.40 (m, 1H), 4.42 (m, 1H), 3.78 (dd, J=12.8, 9.8 Hz, 1H), 3.55 (dd,J=13.1, 9.7 Hz, 1H), 2.20-2.05 (m, 2H) ppm. ³¹P NMR (D₂O, 300 MHz) δ14.66 ppm. MS (m/z): calculated 306.05 (M−H⁻), found 305.8 (M−H⁻).

Example 245 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 543-5: To phosphonic diacid 542-4 (9 mg, 0.029mmol) in 0.25 mL of DMF was added tributylamine (5.4 mg, 0.03 mmol)followed by carbonyldiimidazole (48 mg, 0.3 mmol). Reaction mixture wasstirred at room temperature for 4 h at which point MeOH (0.010 mL) wasadded and stirred for an additional 30 min. Tributyl ammoniumpyrophosphate (161 mg, 0.3 mmol) in DMF (0.64 mL) was added the reactionmixture stirred for 14 h. After the solvents were evaporated in vacuo,the crude product was purified by ion exchange HPLC (0-40% TEAB) to givethe triethylammonium salt of 543-5 as a white solid. Yield: 3 mg. ¹H NMR(D₂O, 300 MHz) δ 7.78 (d, J=7.6 Hz, 1H), 6.02 (d, J=7.6 Hz, 1H), 5.88(d, J=2.9 Hz, 1H), 5.42 (m, 1H), 4.41 (m, 1H), 4.05-3.62 (m, 2H), 3.05(q, J=7.4 Hz, triethylammonium), 2.23-1.95 (m, 2H), 1.13 (t, J=7.4 Hz,triethylammonium) ppm. ³¹P NMR (D₂O, 300 MHz) δ 7.58 (d), −8.34 (d),−22.71 (t) ppm. MS (m/z): calculated 465.98 (M−H⁻), found 466.16 (M−H⁻).

Example 246 Synthesis of Exemplary Compounds of the Invention

Synthesis of 3′-Deoxy-CP

Example 247 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 545-2 To a stirred solution of1-O-methyl-2′-deoxy-D-ribose (23.9 g, 161.41 mmol) in pyridine undernitrogen was added t-butyldiphenylsilyl chloride (48 mL, 186 mmol)dropwise. When the addition was complete N,N-dimethyl-4-aminopyridinewas added as a solid. The reaction was stirred at room temperature for12 hr. and monitored by TLC. When the reaction was complete by TLC thepyridine was removed under vacuum. The oily residue was suspended inethyl acetate (150 mL) and a white solid formed. The mixture wasfiltered and the solid was washed with 50 mL additional ethyl acetate.The solid was then discarded. The organic filtrates were combined andwashed with water (2×100 mL), 1N HCl (aq) (2×100 mL) and sodiumbicarbonate (sat'd) (2×100 mL). The organic phase was collected anddried over MgSO₄(anh). Evaporation and purification by columnchromatography provides the desired mixture of diastereomers 545-2:yield 31.15 g (50 0%). ¹H NMR (CD₃CN, 300 MHz): δ 1.08 m, 9H); 1.85 m1H, 2.27 m 2H, 3.3 s 3H, 3.7 m 2H, 3.90 m 1H, 4.27 m 1H, 5.06 m 1H, 7.45m 6H, 7.76 m 4H. ppm.

Example 248 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 546-3: The alcohol 545-2 (5.00 g, 12.95 mmol) andtriphenylphosphine (6.79 g. 25.9 mmol) were dissolved in anhydrous THF(50 mL) under nitrogen at room temperature. To this stirring solutionwas added dropwise, a mixture of benzoic acid (3.162 g, 25.9 mmol) anddisopropylazodicarboxylate dissolved in anhydrous THF (30 mL). After theaddition was complete the reaction was stirred for 12 hr. at roomtemperature. After the reaction was complete by TLC, the solvent wasremoved under vacuum. The residue was suspended in diethyl ether (60mL). Hexane (120 mL) was added and the solid formed was filtered anddiscarded. The solvents were removed by rotary evaporation and theproducts 546-3 were purified by column chromatography (2% to 15% EtOAcin hexane): yield 3.074 g (48.4%). ¹H NMR (CD₃CN, 300 MHz): δ 0.98 m 9H,2.07 m 1H, 2.42 m 2H, 3.35 s 3H, 3.85 m 1H, 3.99 m 1H, 4.4 m 1H, 5.10 m1H, 5.69 m 1H, 7.30 m 1H, 7.47 m 5H, 7.65 m 6H, 7.80 m 1H, 7.95 m 1H,8.22 m 1H ppm.

Example 249 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 547-4: The acetal 546-3 (6.88 g, 12.95 mmol) andhydroxymethylphosphonate diethyl ester (7.76 mL, 52.65 mmol) weredissolved in 200 mL of toluene. The toluene was removed by rotaryevaporation at 70° C. under vacuum to reduce the reaction volume toapproximately 25 mL. The reaction was cooled to room temperature andp-toluene sulfonic acid monohydrate (0.490 g, 2.58 mmol) was added as asolid along with toluene (200 mL). The toluene was removed by rotaryevaporation at 70° C. under vacuum to reduce the reaction volume onceagain to approximately 25 mL. Two additional aliquots of toluene wereadded and removal by evaporation is repeated each time. The reaction wasmonitored by TLC and when completed the residue was suspended in ethylacetate (100 mL) The organic layer was washed with sodium bicarbonate(sat'd), brine and then dried over MgSO₄ (anh). The desired phosphonate547-4 was purified by column chromatography (10% to 90% EtOAc inhexane): yield 2.89 g (33%). ¹H NMR (DMSO-d₆, 300 MHz): δ 0.92 s 9H,1.25 t 6H, 2.35 t 2H, 3.84 m 4H, 4.05 m 4H, 4.32 q 1H, 5.37 t 1H, 5.62 q1H, 7.26 t 2H, 7.30-7.55 m 8H, 7.60 d 2H, 7.65 t 1H; 7.82 d 2H ppm.

Example 250 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 548-5: The silyl ether 547-4 (2.86 g, 4.57 mmol)was dissolved in a minimal amount of methanol (15 mL) and stirred a roomtemperature under nitrogen. Ammonium fluoride (1.69 g, 45.7 mmol) wasadded as a solid and the reaction was stirred at room temperature for 12hr. The reaction was monitored by TLC and, when complete the methanolwas removed under a stream of nitrogen. Add 6 mL of 1N Acetic acid (aq)and extract the aqueous phase with ethyl acetate (2×125 mL). Combine theorganic extracts and dried over Na₂SO₄(anh). The final product 548-5 waspurified by column chromatography (50% to 100% EtOAc in hexane): yield1.59 g (90%). ¹H NMR (DMSO-d₆, 300 MHz): δ 1.21 t 6H, 2.35 t 2H,3.62-3.82 m 4H, 4.05 m 4H, 4.12 q 1H, 5.30 t 1H, 5.49 q 1H, 7.47 t 2H,7.65 t 1H; 7.90 d 2H.

Example 251 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 549-6: The primary alcohol 548-5 (1.43 g, 3.69mmol) was dissolved in a 1:1 mixture of acetonitrile and water (10 mL)under nitrogen. Bisacetyliodobenzene (2.61 g, 8.12 mmol) was added as asolid along with a catalytic amount of TEMPO (0.115 g, 0.74 mmol). Thereaction was stirred at room temperature for 12 hr. and monitored byTLC. When the reaction was complete, it was frozen and lyophilized. Thecarboxylic acid 549-6 was purified by column chromatography (0% to 10%methanol in dichloromethane): yield 0.750 g (51%). ¹H NMR (CD₃CN, 300MHz): δ 1.30 t 6H, 2.45 t 2H, 3.84 m 1H, 4.00-4.20 m 5H, 4.82 d 1H, 5.50t 1H, 5.82 q 1H, 7.54 t 2H, 7.65 t 1H, 7.96 d 2H.

Example 252 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 550-7 To acid 549-6 (88 mg, 0.22 mmol) in DMF (3.1mL, 0.07 M) was added anhydrous pyridine (0.027 mL, 0.33 mmol) followedby lead tetraacetate (146 mg, 0.33 mmol). After 14 h at roomtemperature, Et₂O/H₂O (1:1, 3 mL) was added. The organics wereseparated, washed with 1M aqueous citric acid, saturated aqueous NaHCO₃,saturated aqueous NaCl and dried over sodium sulfate. After removal ofsolvent, the crude product 7 (50 mg, 54%) was used directly in the nextreaction.

Example 253 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 551-8: N-Acetoxy-diphenylcarbamoyl gaunine (43 mg,0.11 mmol), synthesized as described in Can. J. Chem. 65: 1436 (1987),in dichloroethane (1.1 mL, 0.1 M) was treated withN,O-bis(trimethylsilyl)acetamide (0.054 mL, 0.22 mmol). The reactionmixture was heated to 80° C. for 20 min after which the solvents wereremoved in vacuo. The crude silylated protected guanine was combinedwith phosphonate 550-7 (50 mg, 0.12 mmol) in dichloroethane (1.1 mL, 0.1M) to which TMSOTf (28, 0.153 mmol) was added. The reaction mixtureheated to 60° C. for 5 h after with the reaction was quenched withsaturated aqueous NaHCO₃. The solution was extracted with CH₂Cl₂, washedwith saturated aqueous NaHCO₃, and dried over sodium sulfate. Afterremoval of solvent, the crude product was purified by columnchromatography on silica (2% MeOH/CH₂Cl₂) to provide the phosphonatediester 551-8 (18 mg, 22%). ¹H NMR (CDCl₃, 300 MHz) δ 8.30 (s, 1H), 8.15(s, 1H), 8.02 (s, 1H) 8.00 (s, 1H), 7.35-7.62 (m, 12H), 6.43 (d, 1H),6.02 (m, 1H), 5.65 (m, 1H), 4.18 (q, 4H), 3.78-4.01 (m, 2H), 2.86 (m,1H), 2.63 (m, 1H), 2.53 (s, 3H), 1.37 (t, 6H) ppm. ³¹P NMR (CDCl₃, 300MHz) δ 20.07 (s) ppm. MS (m/z): calculated 744.2 (M+H⁺), found 744.9(M+H⁺).

Example 254 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 552-9 Phosphonate ester 551-8 (14 mg, 0.02 mmol)was treated with NH₃ in MeOH (2 mL, 2.0 N) at room temperature for 9 h.After solvents were removed in vacuo, the crude product was purified bycolumn chromatography on silica (10% MeOH/CH₂Cl₂) to provide 552-9. ¹HNMR (CD₃OD, 300 MHz) δ 7.89 (s, 1H), 5.96 (d, 1H), 5.45 (m, 1H),4.10-4.21 (q, 4H), 3.84-4.02 (m, 2H), 2.92-2.47 (m, 2H), 1.33 (t, 6H)ppm. ³¹P NMR (CD₃OD, 300 MHz) δ 21.75 ppm. MS (m/z): calculated 404.1(M+H⁺), found 404.2 (M+H⁺).

Example 255 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 553-10: To phosphonate ester guanosine derivative552-9 (5.8 mg, 0.02 mmol) in anhydrous acetonitrile (0.15 mL, 0.1 M) wasadded 2,6-lutidine (0.014 mL, 0.12 mmol) followed by iodotrimethylsilane(0.016 mL, 0.12 mmol). After stirring for 15 min, triethylamine (0.12mmol) and methanol (0.020 mL) were added and solvents were removed invacuo. The crude product was purified by reverse phase columnchromatography on C18 (0-10% MeOH/H₂O-1% AcOH) to provide the phosphonicdiacid 553-10. ¹H NMR (D₂O, 300 MHz) δ 7.91 (s, 1H), 5.86 (d, 1H), 5.41(m, 1H), 3.42-3.65 (m, 2H), 2.25-2.36 (m, 2H) ppm. ³¹P NMR (D₂O, 300MHz) δ 15.16 ppm. MS (m/z): calculated 346.1 (M−H⁻), found 346.3 (M−H⁻).

Example 256 Synthesis of Exemplary Compounds of the Invention

Synthesis of Compound 554-11: To phosphonic diacid 553-10 (2.5 mg, 7.2μmol) in DMF (144 μL, 0.05 M) was added tributylamine (0.0086 mL, 0.036mmol) followed by carbonyldiimidazole (12 mg, 0.072 mmol). Reaction wasstirred at room temperature for 12 h at which point MeOH (0.005 mL) wasadded and stirred for an additional 30 min. Tributyl ammoniumpyrophosphate (0.040 mg, 72 mmol) in DMF (0.16 mL) was added thereaction mixture stirred for 1 h. After the solvents were evaporated invacuo, the crude product was purified by ion exchange HPLC (0-60% TEAB)to provide the diphosphophosphonate 554-11. ¹H NMR (D₂O, 300 MHz) δ 7.94(s, 1H), 5.85 (d, 1H), 4.47 (m, 1H), 3.71-3.78 (m, 2H), 2.27-2.39 (m,2H) ppm. ³¹P NMR (D2O, 300 MHz) δ 8.09 (d), 7.71 (s), −22.04 (t) ppm. MS(m/z): calculated 505.99 (M−H⁻), found 506.2 (M−H⁻).

Example 257 Synthesis of Exemplary Compounds of the Invention

The following Schemes 555-5 to 555-9 describes a general method ofpreparing the [3.1.0] bicyclo hexane scaffold of the Formula 555-I and555-II compounds. Exemplary structures, intermediates, substituents,protecting groups, reagents, and synthetic routes chosen for descriptionhere are meant to merely illustrate general methods of preparation, andare not intended to any way limit or denote preference to the methods.

The [3.1.0] bicyclo hexane scaffold may be synthesized by intramolecularcyclopropanation of a carbene generated by decomposition of a diazo1,3-ketoester (Moon et al (2000) Organic Letters 2(24):3793-3796). Therequisite 1,3-ketoester 555-5.1 may be prepared from acetoacetate esteranion addition to ene-aldehydes, e.g. acrolein (Scheme 555-5a). Forexample, ethyl acetoacetate treated with 2 equivalents of lithiumdiisopropylamide at −78° C. and then one equivalent of acrolein givesthe 1,3-ketoester 555-5.1 (Yoshimura et al (2002) Jour. Org. Chem.67:5938-5945). Protection of the hydroxyl may be accomplished withphenyldimethylsilyl chloride to give 555-5.2 where PG isphenyldimethylsilyl (PhMe₂Si—). Other trialkylsilyl protecting groupsmay be useful. Diazotization of 2 with p-toluenesulfonyl azide gives555-5.3. Treatment of 555-5.3 with a carbenoid insertion catalyst, e.g.CuSO₄ or Rh(OAc)₂, gives 555-5.4 as a mixture of diastereomers.

The ester of 555-5.4 may be hydrolyzed to the hydroxymethyl 555-5.5 orsaponified directly to carboxylic acid 555-5.6 (Scheme 555-5b).Appropriate oxidant(s) can convert the primary alcohol 555-5.5 tocarboxylic acid 555-5.6 or its corresponding ester. In the case of anester, an additional deprotection step will give the carboxylic acid,555-5.6. A variety of oxidation procedures exist in the literature andcan be utilized here. These include but are not limited to the followingmethods: (i) pyridinium dichromate in Ac₂O, t-BuOH, and dichloromethaneproducing the t-butyl ester, followed by a deprotection using reagentsuch as trifluoroacetic acid to convert the ester to the correspondingcarboxylic acid (see Classon, et al, Acta Chem. Scand. Ser. B; 39; 1985;501-504. Cristalli, et al; J. Med. Chem.; 31; 1988; 1179-1183); (ii)iodobenzene diacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy, freeradical (TEMPO) in acetonitrile, producing the carboxylic acid (See Epp,et al; J. Org. Chem. 64; 1999; 293-295. Jung et al; J. Org. Chem.; 66;2001; 2624-2635.); (iii) sodium periodate, ruthenium(III) chloride inchloroform producing the carboxylic acid (see Kim, et al, J. Med. Chem.37; 1994; 4020-4030. Homma, et al; J. Med. Chem.; 35; 1992; 2881-2890);(iv) chromium trioxide in acetic acid producing the carboxylic acid (seeOlsson et al; J. Med. Chem.; 29; 1986; 1683-1689. Gallo-Rodriguez et al;J. Med. Chem.; 37; 1994; 636-646); (v) potassium permanganate in aqueouspotassium hydroxide producing the carboxylic acid (see Ha, et al; J.Med. Chem.; 29; 1986; 1683-1689. Franchetti, et al; J. Med. Chem.; 41;1998; 1708-1715.) (vi) nucleoside oxidase from S. maltophilia to givethe carboxylic acid (see Mahmoudian, et al; Tetrahedron; 54; 1998;8171-8182.)

Carboxylic acid 555-5.6 may be converted by decarboxylation to acetate555-5.7 using lead(IV) tetraacetate (Teng et al; (1994) J. Org. Chem.;59:278-280; Schultz, et al; J. Org. Chem.; 48; 1983; 3408-3412. Whenlead(IV) tetraacetate is used together with lithium chloride (see Kochi,et al; J. Am. Chem. Soc.; 87; 1965; 2052), the corresponding chloride isobtained 555-5.8. Lead(IV) tetraacetate in combination withN-chlorosuccinimide can also produce 555-5.8 (Wang, et al; Tet. Asym.;1; 1990; 527 and Wilson et al; Tet. Asym.; 1; 1990; 525). Alternatively,the acetate can also be converted to other leaving groups such asbromide by treatment of trimethylsilyl bromide (Spencer, et al; J. Org.Chem.; 64; 1999; 3987-3995).

Intermediates 555-5.7 and 555-5.8 may react with a variety ofnucleophiles as described by Teng et al; Synlett; 1996; 346-348 and U.S.Pat. No. 6,087,482; Column 54 line 64 to Column 55 line 20.Specifically, 555-5.7 may be reacted with diethylhydroxymethylphosphonate in the presence of trimethylsilyltrifluoromethanesulfonate (TMS-OTf) to give 555-5.9 (Scheme 555-5c). Itcan be envisioned that other compounds with the general structure ofHO-linker-POR^(P1)R^(P2) can also be used so long as the functionalgroups in these compounds are compatible with the coupling reactionconditions. There are many examples in the published literaturedescribing the coupling of 1′ acetyl furanosyl compounds with a varietyof alcohols. The reactions can be facilitated with a number of reagents,such as silver(I) salts (see Kim et al (1991) J. Org. Chem.56:2642-2647, Toikka et al (1999) J. Chem. Soc. Perkins Trans. 1;13:1877-1884); mercury(II) salts (see Veeneman et al (1987) Recl. Trav.Chim. Pays-Bas; 106:129-131); boron trifluoride diethyl etherate (seeKunz et al (1985) Hel. Chim Acta; 68:283-287); tin(II) chloride (seeO'Leary et al (1994) J. Org. Chem. 59:6629-6636); alkoxide (seeShortnacy-Fowler et al (2001) Nucleosides & Nucleotides; 20:1583-1598);and iodine (see Kartha et al (2001) J. Chem. Soc. Perkins Trans.1770-772). These methods can be selectively used in conjunction withdifferent methods in forming intermediates from 555-5.6 with variousleaving groups (LG).

The introduction and removal of protecting groups (represented in thestructures herein as PG) from a compound is common practice art inorganic synthesis. Many sources of information of the art are availablein the published literature, e.g. Greene and Wuts, Protecting Groups inOrganic Synthesis, 3^(rd) Ed., John Wiley & Sons, Inc., 1999. The mainpurpose is to temporarily transform a functional group and mask itsreactivity so that it will survive a set of subsequent reactionprocedures. Afterwards, the original functional group can be restored bya preconceived deprotection procedure. Therefore, the transformations inSchemes 555-(5a-c) are intended to build the [3.1.0] scaffold with theappropriate latent functionality or reactivity components.

The keto group of certain intermediates, e.g. 555-5.4 may be elaboratedto ribofuranose-type analogs 555-6.1 where Z¹ are for example, eachhydroxyl or protected hydroxyl (Scheme 555-6). The hydroxyl groups canbe protected as benzoyl (Bz) esters to give 555-6.2. The bridgeheadcarboxylate ester can then be orthogonally hydrolyzed to give 555-6.3 orreduced to hydroxymethyl 555-6.4. Oxidation of 555-6.4, e.g. usingiodobenzene diacetate and 2,2,6,6-tetramethyl-1-piperidinyloxy, freeradical (TEMPO), converts the primary alcohol to the corresponding acid555-6.3. Further oxidation of 555-6.3 using lead tetraacetate canproduce acetate 555-6.5. Coupling between 555-6.5 and hydroxyalkyldialkylphosphonate compounds, e.g. diethyl hydroxymethylphosphonate(available from Sigma-Aldrich, Cat. No. 39, 262-6) and TMS-OTf canafford 555-6.6. Treating 555-6.6 with TMS-Br converts the phosphodiesterto the corresponding phosphonic acid 555-6.7. Deprotection, e.g. NH₃ inmethanol, of the 2′- and 3′-hydroxyl gives 555-6.8.

The phosphonic acids in 555-(6.6-6.8) are used as examples forillustration purpose. Other forms of phosphonates can be accessed viathe phosphonic acid, or other forms, such as the corresponding diesters.See Schemes 555-A and 555-(1-4) for exemplary interconversions ofphosphonate moieties.

Compounds such as 555-6.6 can be further elaborated by selectivedeprotection of PG and introduction of the nucleobase moiety (B). Forexample, where PG is trialkylsilyl, e.g. triethylsilyl,t-butyldimethylsilyl, or phenyldimethylsilyl, treatment of 555-6.6 witha fluoride reagent, e.g. tetrabutylammonium fluoride in THF, mayselectively remove PG. The resulting hydroxyl may be converted to aleaving group (LG) such as chloro or acetate 555-7.1 underVorbruggen-type reaction conditions, or the hydroxyl 555-7.2 reacted insitu, e.g. Mitsunobu conditions, to establish the carbon-nitrogen bondwith a nucleobase or protected nucleobase reagent to give 555-7.3(Scheme 555-7). Suitable nucleobase or protected nucleobase reagents (B)include thymidine, cytosine, adenine, guanine, and silylated formsthereof. The resulting covalent attachment may be 9-purinyl or1-pyrimidinyl. Other positional isomers may result and conventionalmeans of separation may be employed to generate pure 555-7.3 compounds.The 2′ and 3′ protecting groups (Bz=benzoyl) may be removed fromintermediate 555-7.3 with aqueous base to give 555-7.4. The ethyl groupsof 555-7.4 may be removed with a dealkylation reagent such astrimethylsilyl bromide to give phosphonic acid 555-7.5 which may befurther elaborated according to the reactions shown in Schemes 555-A and1-4 to other phosphonate moieties, including diphosphophosphonate andphosphophosphonate compounds.

Scheme 555-8 shows an exemplary route to 2′-β-methyl, 2′-3′ hydroxylbicylco adenine compounds. The 3′ and 5′ hydroxyl groups of [3.1.0]bicyclo analog of adenosine 555-8.1 (Kim, et al (2002) J. Med. Chem.45:208-218) may be selectively silylated to give 555-8.2. The 2′hydroxyl group may be oxidized under Dess-Martin periodinane conditionsto give 555-8.3. The 2′ keto of 555-8.3 may be methylenated with aWittig reagent and desilylated to give 555-8.4. Epoxidation of 555-8.4gives 555-8.5. Hydride attack on the methylene carbon of the epoxide555-8.5 gives 555-8.6 with the 2′,3′-α-dihydroxy, 2′-β-methyl motif.This synthetic route may be versatile in the preparation of a variety of2′,3′-α-dihydroxy, 2′-β-methyl[3.1.0] bicyclo compounds where B=anyprotected or unprotected nucleobase.

The 5′ hydroxymethyl group of 2′,3′-α-dihydroxy, 2′-β-methyl [3.1.0]bicyclo compounds, e.g. 555-8.6, may be elaborated by selectivereactions such as those shown in Scheme 555-9. The 5′ carbon may beremoved by an oxidative decarboxylation to allow attachment of aphosphonate moiety through an oxygen atom bound directly to the [3.1.0]scaffold. The 5′ hydroxyl group of 555-8.6 may be selectively protectedas the 5′ tert-butyldiphenylsilyl ether (TBDPS) and then the 2′ and 3′hydroxyls may be protected as methoxymethyl ethers to give 555-9.1. The5′ TBDPSi group may be removed with tetra-butyl ammonium fluoride (TBAF)and the resulting hydroxymethyll oxidized with the periodinane reagent,PhI(OAc)₂ and TEMPO to the carboxylic acid 555-9.2. Oxidativedecarboxylation of 555-9.2 with lead tetracetate and treatment withlithium hydroxide gives 555-9.3. Alkylation of the hydroxyl of 555-9.3with bromomethyldiethyl phosphonate gives 555-9.4. The phosphonate ethylgroups and the 2′,3′ methoxymethyl (MOM) protecting groups may beremoved with iodotrimethylsilane to give 555-9.5. The phosphonic acidgroup of 555-9.5 may be activated, for example with carbonyldiimidazole(CDI) and reacted with pyrophosphate anion to give diphosphophosphonate555-9.6. Other phosphonic acid conversions may be conducted, asdescribed in Schemes 555-A and 555(1-4).

Intermediate 555-8.2 is versatile for the preparation of 2′ hydroxyl[3.1.0] bicyclo compounds (Scheme 555-10). Protection of the 2′ hydroxylwith para-methoxybenzyl bromide and silyl removal with TBAF gives555-10.1. The 5′ hydroxyl is protected with the TBDPSi group to give555-10.2. The 3′ thioester of 555-10.2 is formed with phenylchlorothionoformate followed by reduction with tributyl tin hydride withAIBN to give 555-10.3.. Desilyation with TBAF and oxidation with BAIBand TEMPO furnishes the carboxylic acid 555-10.4.

Oxidative decarboxylation of 555-10.4 with lead tetracetate followed bylithium hydroxide treatment gives the hydroxyl 555-11.1 (Scheme 555-11).Alkylation of hydroxyl 555-11.1 with bromomethyl diethyl phosphonategives 555-11.2. Iodotrimethylsilane cleaves the ethyl groups from thediethylphosphonate and ceric ammonium nitrate deprotects thepara-methoxy benzyl group of 555-11.2 to give 555-11.3. Phosphonateactivation with CDI and addition of pyrophosphate gives the 2′-hydroxydiphosphophosphonate [3.1.0] compound 555-11.4.

Example 258 Synthesis of Exemplary Compounds of the Invention

The following Schemes describe the general method of preparing the 2′fluoro, 2′-3′ didehydro nucleosidescaffold of compounds of the presentinvention.

Methods of introduction of fluorine at the 2′ position ofribonucleosides and nucleoside analogs are described in U.S. Pat. No.5,824,793; U.S. Pat. No. 5,859,233; Choo, H. et al Journal of MedicinalChemistry (2003), 46(3), 389-398; Moon, H. et al Journal of the ChemicalSociety, Perkin Transactions 1 (2002), (15), 1800-1804; Lee, Kyeong;Choi, Y. et al Journal of Medicinal Chemistry (2002), 45(6), 1313-1320;Lee, Kyeong; Choi, Yongseok; Hong, J. et al Nucleosides & Nucleotides(1999), 18(4 & 5), 537-540; Lee, K. et al Journal of Medicinal Chemistry(1999), 42(7), 1320-1328; Choi, Y. et al Tetrahedron Letters (1998),39(25), 4437-4440; Chen, Shu-Hui et al Bioorganic & Medicinal ChemistryLetters (1998), 8(13), 1589-1594; Siddiqui, Maqbool et al TetrahedronLetters (1998), 39(13), 1657-1660; Nakayama, Toshiaki et al NucleicAcids Symposium Series (1991), 25 (Symp. Nucleic Acids Chem., 18th,1991), 191-2; Huang, Jai Tung et al Journal of Medicinal Chemistry(1991), 34(5), 1640-6; Sterzycki, Roman Z et al Journal of MedicinalChemistry (1990), 33(8), 2150-7; Martin, Joseph A et al Journal ofMedicinal Chemistry (1990), 33(8), 2137-45; Watanabe, Kyoichi et alJournal of Medicinal Chemistry (1990), 33(8), 2145-50; Zemlicka et alJournal of the American Chemical Society (1972) 94(9):3213-3218.

Schemes 556 (A-F) show the synthetic routes which have been utilized toprepare the exemplary embodiments shown therein.

The (−) enantiomer of adenosine 556-A.1 was tritylated at the N-6 of theadenine exocyclic amine and the 5′ hydroxyl with an excess of trityl(Tr, triphenylmethyl, Ph₃C—) chloride with dimethylaminopyridine inpyridine to give bis-trityl 556-A.2 which was treated with triflicanhydride in dichloromethane and DMAP to give 556-A.3 (Scheme 556-A).The 2′ triflate group was displaced by fluoride with tetra-butylammoniumfluoride in THF at room temperature to give 556-A.4.

Example 259

By way of example and not limitation, embodiments of the invention arenamed below in tabular format (Table 100). These embodiments are of thegeneral formula “MBF”:

Each embodiment of MBF is depicted as a substituted nucleus (Sc). Sc isdescribed in formula 1-108 herein, wherein A⁰ is the point of covalentattachment of Sc to Lg, as well as in Tables 1.1 to 1.5 below. For thoseembodiments described in Table 100, Sc is a nucleus designated by anumber and each substituent is designated in order by letter or number.Tables 1.1 to 1.5 are a schedule of nuclei used in forming theembodiments of Table 100. Each nucleus (Sc) is given a numberdesignation from Tables 1.1 to 1.5, and this designation appears firstin each embodiment name. Similarly, Tables 10.1 to 10.19 and 20.1 to20.36 list the selected linking groups (Lg) and prodrug (Pd¹ and Pd²)substituents, again by letter or number designation, respectively.Accordingly, a compound of the formula MBF includes compounds having Scgroups based on formula 1-108 herein as well as compounds according toTable 100 below. In all cases, compounds of the formula MBF have groupsLg, Pd¹ and Pd² set forth in the Tables below.

Accordingly, each named embodiment of Table 100 is depicted by a numberdesignating the nucleus from Table 1.1-1.5, followed by a letterdesignating the linking group (Lg) from Table 10.1-10.19, and twonumbers designating the two prodrug groups (Pd¹ and Pd²) from Table20.1-20.36. In graphical tabular form, each embodiment of Table 100appears as a name having the syntax:

Sc.Lg.Pd¹.Pd²

Each Sc group is shown having a tilda (“˜”). The tilda is the point ofcovalent attachment of Sc to Lg. Q¹ and Q² of the linking groups (Lg),it should be understood, do not represent groups or atoms but are simplyconnectivity designations. Q¹ is the site of the covalent bond to thenucleus (Sc) and Q² is the site of the covalent bond to the phosphorousatom of formula MBF. Each prodrug group (Pd¹ and Pd²) are covalentlybonded to the phosphorous atom of MBF at the tilda symbol (“˜”). Someembodiments of Tables 10.1-10.19 and 20.1-20.36 may be designated as acombination of letters and numbers (Table 10.1-10.19) or number andletter (Table 20.1-20.36). For example there are Table 10 entries forBJ1 and BJ2. In any event, entries of Table 10.1-10.19 always begin witha letter and those of Table 20.1-20.36 always begin with a number. Whena nucleus (Sc) is shown enclosed within square brackets (“[ ]”) and acovalent bond extends outside the brackets, the point of covalentattachment of Sc to Lg may be at any substitutable site on SC. Selectionof the point of attachment is described herein. By way of example andnot limitation, the point of attachment is selected from those depictedin the schemes and examples.

Lengthy table referenced here US20090275535A1-20091105-T00001 Pleaserefer to the end of the specification for access instructions.

All literature and patent citations above are hereby expresslyincorporated by reference at the locations of their citation.Specifically cited sections or pages of the above cited works areincorporated by reference with specificity. The invention has beendescribed in detail sufficient to allow one of ordinary skill in the artto make and use the subject matter of the following Claims. It isapparent that certain modifications of the methods and compositions ofthe following Claims can be made within the scope and spirit of theinvention.

In the claims hereinbelow, the subscript and superscripts of a givenvariable are distinct. For example, R₁ is distinct from R¹.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090275535A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A conjugate of the following formula:

or a pharmaceutically acceptable salt or solvate thereof; wherein: B isselected from adenine, guanine, cytosine, uracil, thymine,7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine,7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine,isocytosine, isoguanine, 7-deazaguanine, 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, O⁶-methylguanine,N⁶-methyladenine, O⁴-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, substituted triazole, andpyrazolo[3,4-d]pyrimidine; X is selected from O, C(R₁)₂, OC(R^(y))₂, NRand S; Z is independently selected from H, OH, OR, NR₂, CN, NO₂, SH, SR,F, Cl, Br, and I; Y¹ is independently O, S, NR, ⁺N(O)(R), N(OR),⁺N(O)(OR), or N—NR₂; Y² is independently O, CR₂, NR, ⁺N(O)(R), N(OR),⁺N(O)(OR), N—NR₂, S, S—S, S(O), or S(O)₂; M2 is 0, 1 or 2; R^(y) isindependently H, F, Cl, Br, I, OH, —C(═Y¹)R, —C(═Y¹)OR, —C(═Y¹)N(R)₂,—N(R)₂, —⁺N(R)₃, —SR, —S(O)R, —S(O)₂R, —S(O)(OR), —S(O)₂(OR), —OC(═Y¹)R,—OC(═Y¹)OR, —OC(═Y¹)(N(R)₂), —SC(═Y¹)R, —SC(═Y¹)OR, —SC(═Y¹)(N(R)₂),—N(R)C(═Y¹)R, —N(R)C(═Y¹)OR, or —N(R)C(═Y¹)N(R)₂, amino (—NH₂), ammonium(—NH₃ ⁺), alkylamino, dialkylamino, trialkylammonium, C₁-C₈ alkyl, C₁-C₈alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, 5-7 memberedring sultam, C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino,4-dialkylaminopyridinium, C₁-C₈ alkylhydroxyl, C₁-C₈ alkylthiol,alkylsulfone (—SO₂R), arylsulfone (—SO₂Ar), arylsulfoxide (—SOAr),arylthio (—SAr), sulfonamide (—SO₂NR₂), alkylsulfoxide (—SOR), ester(—C(═O)OR), amido (—C(═O)NR₂), 5-7 membered ring lactam, 5-7 memberedring lactone, nitrile (—CN), azido (—N₃), nitro (—NO₂), C₁-C₈ alkoxy(—OR), C₁-C₈ alkyl, C₁-C₈ substituted alkyl, C₁-C₈ alkenyl, C₁-C₈substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈ substituted alkynyl, C₆-C₂₀aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀ heterocycle, C₂-C₂₀ substitutedheterocycle, polyethyleneoxy, or W³; or when taken together, R^(y) formsa carbocyclic ring of 3 to 7 carbon atoms; R^(x) is independently R^(y),a protecting group, or the formula:

wherein: M1a, M1c, and M1d are independently 0 or 1; M12c is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12; and R is C₁-C₈ alkyl, C₁-C₈ substitutedalkyl, C₁-C₈ alkenyl, C₁-C₈ substituted alkenyl, C₁-C₈ alkynyl, C₁-C₈substituted alkynyl, C₆-C₂₀ aryl, C₆-C₂₀ substituted aryl, C₂-C₂₀heterocycle, C₂-C₂₀ substituted heterocycle, or a protecting group; andW³ is W⁴ or W⁵, where W⁴ is R, —C(Y¹)R^(y), —C(Y¹)W⁵, —SO₂R^(y), or—SO₂W⁵; and W⁵ is a carbocycle or a heterocycle wherein W⁵ isindependently substituted with 0 to 3 R^(y) groups.
 2. The conjugate ofclaim 1 wherein C₁-C₈ substituted alkyl, C₁-C₈ substituted alkenyl,C₁-C₈ substituted alkynyl, C₆-C₂₀ substituted aryl, and C₂-C₂₀substituted heterocycle are independently substituted with one or moresubstituents selected from F, Cl, Br, I, OH, —NH₂, —NH₃ ⁺, —NHR, —NR₂,—NR₃ ⁺, C₁-C₈ alkylhalide, carboxylate, sulfate, sulfamate, sulfonate,5-7 membered ring sultam, C₁-C₈ alkylsulfonate, C₁-C₈ alkylamino,4-dialkylaminopyridinium, C₁-C₈ alkylhydroxyl, C₁-C₈ alkylthiol, —SO₂R,—SO₂Ar, —SOAr, —SAr, —SO₂NR₂, —SOR, —CO₂R, —C(═O)NR₂, 5-7 membered ringlactam, 5-7 membered ring lactone, —CN, —N₃, —NO₂, C₁-C₈ alkoxy, C₁-C₈trifluoroalkyl, C₁-C₈ alkyl, C₃-C₁₂ carbocycle, C₆-C₂₀ aryl, C₂-C₂₀heterocycle, polyethyleneoxy, phosphonate, phosphate, and a prodrugmoiety.
 3. The conjugate of claim 1 wherein protecting group is selectedfrom a carboxyl ester, a carboxamide, an aryl ether, an alkyl ether, atrialkylsilyl ether, a sulfonic acid ester, a carbonate, and acarbamate.
 4. The conjugate of claim 1 wherein W⁵ is selected from thestructures:


5. The conjugate of claim 1 wherein X is O and each R^(y) is H.
 6. Theconjugate of claim 1 wherein the conjugate is a resolved enantiomerhaving the structure:


7. The conjugate of claim 1 wherein the conjugate is a resolvedenantiomer having the structure:


8. The conjugate of claim 1 having the structure:


9. The conjugate of claim 1 having the structure:


10. The conjugate of claim 1 having the structure:


11. The conjugate of claim 1 having the structure:


12. The conjugate of claim 1 having the structure:


13. The conjugate of claim 1 having the structure:

wherein R² is H or C₁-C₈ alkyl.
 14. The conjugate of claim 1 having thestructure:


15. The conjugate of claim 1 having the structure:


16. The conjugate of claim 14 wherein Z is H.
 17. The conjugate of claim14 wherein B is adenine.
 18. The conjugate of claim 14 having thestructure:

wherein Y^(2c) is O, N(R^(y)) or S.
 19. The conjugate of claim 18 havingthe structure:


20. The conjugate of claim 19 wherein Y^(2c) is O.
 21. The conjugate ofclaim 19 wherein Y^(2c) is N(CH₃).
 22. The conjugate of claim 1 whereinsubstituted triazole has the structure:


23. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and a conjugate as described in claim 1, or apharmaceutically acceptable salt or solvate thereof.
 24. A method forpromoting an anti-viral effect in vitro or in vivo comprising contactinga sample in need of such treatment with a conjugate as described inclaim 1, or a pharmaceutically acceptable salt or solvate thereof.
 25. Amethod of inhibiting a viral infection in an animal, comprisingadministering an effective amount of a conjugate as described in claim1, or a pharmaceutically acceptable salt or solvate thereof, to theanimal.
 26. A compound of formula:

or a pharmaceutically acceptable salt or solvate thereof.
 27. Apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a compound of formula:

or a pharmaceutically acceptable salt or solvate thereof.
 28. A methodfor promoting an anti-viral effect in vitro or in vivo comprisingcontacting a sample in need of such treatment with a compound offormula:

or a pharmaceutically acceptable salt or solvate thereof.
 29. A methodof inhibiting a viral infection in an animal, comprising administeringan effective amount of compound of formula:

or a pharmaceutically acceptable salt or solvate thereof, to the animal.